Distillates (petroleum), steam-cracked, C5-12 fraction

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 June 2003 to 27 June 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study available as unpublished report minor restrictions in design and/or reporting but otherwise considered adequate for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material:
not reported

Analytical monitoring:

yes

Details on sampling:

Cell density determined foreach test and control chamber using hemacytometer and microscope at 24, 48, 72 and 96 hrs (± 1 hr). No undissolved test substance observed in test chambers during the study. pH measured on Day 0 and daily after cell density determinations (composite of the three replicates).

Details on test solutions:

Loading Rate (concentration): 0.23 (0.27), 0.46 (0.37), 1.1 (0.94), 2.7 (2.7), 7.3 (7.4) mg/L.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Culture date June 18 2003. Cultured at Environmental Toxicology Laboratory of the testing facility. Initial strain (#1648) provided by UTEX, the Culture Collection of Algae MCDB School of Biological Sciences, the University of Texas at Austin, Austin TX 78712.

Test type:

static

Water media type:

freshwater

Total exposure duration:

96 h

Post exposure observation period:

not reported

Hardness:

not reported

Test temperature:

24.4 ± 0.1 °C

pH:

The pH ranged from 7.5 to 7.6 in the test solutions at test initiation and ranged from 8.2 to 10.2 at test termination.

Dissolved oxygen:

not reported

Salinity:

not reported

Nominal and measured concentrations:

Target Loading Rate* Actual Loading Rate** Measured Concentration†
0 0 0
0.18 0.23 0.27
0.44 0.46 0.37
1.1 1.1 0.94
2.8 2.7 2.7
7 7.3 7.4

* Target loading rate as per protocol.
** Actual loading rate (weight) of test substance added to the vehicle/dilution water.
† Concentration based on mean (Day 0 and Day 4) measured concentrations.

Details on test conditions:

Individual Water Accommodated Fractions (WAFs) were prepared for each treatment. The test substance was added to 4.3 L of algal nutrient medium augmented with sodium bicarbonate in glass aspirator bottles (capacity 4.5 L). The solutions were mixed for approximately 24 hours using an 9% vortex (of the static liquid depth). The test solutions were removed through the outlet at the bottom of each mixing vessel into 12 replicates of 140 mL in 125 mL Erlenmeyer flasks (no headspace) containing two 14 mm glass spheres to facilitate mixing. The test chambers were inoculated with algae (1.0 x 104 cells/mL) and were sealed with ground glass stoppers. Three replicates were sacrificed daily for cell density determination. The test chambers were placed on shaker tables (100 rpm) to keep the algae in suspension. The test was performed under static conditions with no aeration. The algae was cultured in-house from 5 day old stock cultures in log phase growth. Mean test temperature: 24.4°C (sd = 0.1). Continuous light: intensity was 8657 to 8813 Lux. The pH ranged from 7.5 to 7.6 in the test solutions at test initiation and ranged from 8.2 to 10.2 at test termination. Due to the complex nature and limited water solubility of the test substance, the following exceptions to the guideline apply for this study: The concentration of the test substance in solution was not determined prior to use. Test substance analysis was performed on samples of the WAFs at the start of the test (day 0) and at termination (day 4). The initial concentration of the test substance was not maintained at 80% in the three lower loading rates throughout the test (this may be due to biological activity or physical processes in the test chambers). It was appropriate to prepare individual treatment solutions by adding the test substance to dilution water and removing the WAF of each mixture for testing than to prepare dilutions of a stock solution. The test duration was 96 hours, instead of 72 hours. However, both 72 and 96-hour endpoints were determined. None of the above exceptions are believed to have affected the outcome, integrity, or quality of the study.

Reference substance (positive control):

not specified

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

96 h

Dose descriptor:

other: ErC50

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.37 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.94 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Effects on growth rate (r) based upon actual loading rates for Low Dicyclopentadiene Resin Oil (Low DCPD Resin Oil): 72 and 96 hr ErL50 = 1.5 mg/L (<0.23* - >7.3* mg/L) 72 hr NOELR = 0.46 mg/L 96 hr NOELR = 1.1 mg/L (average specific growth rate).
Effects on growth rate (r) based on measured concentrations: 72 and 96 hr ErC50 1.4 mg/L 72 hr NOEC 0.37 mg/L 96 hr NOEC 0.94 mg/L (average specific growth rate).
Effects on biomass (b) based upon actual loading rates; 72 hr EbL50 2.1 mg/L 96 hr EbL50 1.9 mg/L 72 hr NOELR 0.23 mg/L 96 hr NOELR <0.23 mg/L.
Effects on biomass (b) based upon measured concentrations; 72 hr EbC50 2.0 mg/L 96 hr EbC50 1.9 mg/L 72 hr NOEC 0.27 mg/L 96 hr NOEC <0.27 mg/L.

Results with reference substance (positive control):

not reported

Reported statistics and error estimates:

95% confidence intervals

Hours	EL50 mg/L	ErC50 mg/L	EbL50 mg/L	EbC50 mg/L
72	1.5 (<0.23*->7.3*)	1.4 (0.27 - >7.4*)	2.1 (<0.23*->7.3*)	2.0 (<0.27*->7.4*)
96	1.5 (<0.23*->7.3*)	1.4 (<0.27*->7.4*)	1.9 (<0.23*->7.3*)	1.9 (<0.27*->7.4*)

 Values in parentheses ( ) are 95% confidence levels

Hours	Average Specific Growth Rate		Area under the growth curves
	NOELR	NOEC	NOELR	NOEC
72	0.46 mg/L	0.37 mg/L	0.23 mg/L	0.27 mg/L
96	1.1 mg/L	0..94 mg/L	<0.23 mg/L*	<0.27 mg/L*

 * The highest or lowest loading rate or concentration tested

Validity criteria fulfilled:

yes

Conclusions:

The Effects on growth rate (r) based upon actual loading rates for Low Dicyclopentadiene Resin Oil (Low DCPD Resin Oil):
72 and 96 hr ErL50 = 1.5 mg/L (<0.23* - >7.3* mg/L)
72 hr NOELR = 0.46 mg/L
96 hr NOELR = 1.1 mg/L
72 hr NOEC 0.37 mg/L (measured concentrations)
96 hr NOEC 0.94 mg/L (meaured concentrations)

Executive summary:

This is a GLP compliant, guideline study considered adequate for assessment. The effects on growth rate (r) based upon actual loading rates for Low Dicyclopentadiene Resin Oil (Low DCPD Resin Oil):72 and 96 hr E_rL₅₀ = 1.5 mg/L (<0.23* - >7.3* mg/L); 72 hr NOELR = 0.46 mg/L; 96 hr NOELR = 1.1 mg/L; 72 hr NOEC 0.37 mg/L (measured concentrations); 96 hr NOEC 0.94 mg/L (measured concentrations). Whilst this study will fulfil the REACH endpoint and is considered suitable for assessment, as it is based upon a WAF procedure it would not be suitable for deriving a PNEC.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 May 2003 to 24 May 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study available as unpublished report minor restrictions in design and/or reporting but otherwise considered adequate for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material:
Not reported

Analytical monitoring:

yes

Details on sampling:

12 replicates prepared for each treatment and 12 replicates of the control. Cell density determined for each test and control chamber using hemacytometer and microscope at 24, 48, 72 and 96 hrs. Cell density determinations performed on 3 replicates at each observation. pH measured on Day 0 and daily after cell density determinations (composite of the 3 samples).

Details on test solutions:

Individual treatments prepared by adding appropriate amount of test substance to 8.4 L of algal nutrient media in glass aspirator bottles (cap. 8.7L) and stirring on magnetic stirplates using an approx. 9% (of the static liquid depth) vortex for 24 hrs.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Source: cultured at the Environmental Toxicology Laboratory of the testing facility. Initial strain (#1648) provided by UTEX. The culture collection of Algae MCDB School of Biologial Sciences, the Unviersity of Texas at Austin received by the lab on 13 Feb 01. Cultured and tested in aprox. 300mL of nutrient media.
Holding conditions prior to test: Cell counts performed weekly to ensure cells are in log phase of growth and to verify culture is axenic. New culture started weekly using inoculum from previous culture. Held at 22-25 ºC under continuous illumination.
Life stage of test species used: taken from 5 day old stock cultures in log phase of growth.

Test type:

static

Water media type:

freshwater

Total exposure duration:

96 h

Post exposure observation period:

not reported

Hardness:

not reported

Test temperature:

24 degrees C

pH:

ranged from 7.5-7.6 at the beginning of the test and from 8.1-9.8 at the end of the test.

Dissolved oxygen:

not reported

Salinity:

not reported

Nominal and measured concentrations:

Target loading rate* Actual loading rate** Measured concentration†
0 0 0
0.18 0.17 0.14
0.44 0.47 0.3
1.1 1.2 0.83
2.8 2.6 1.9
7 6.9 5.7
* Target loading rate as per protocol.
** Actual loading rate (weight) of test substance added to the vehicle/dilution water.
† Concentration based on mean (Day 0 and Day 4) measured concentrations.

Details on test conditions:

Individual Water Accommodated Fractions (WAFs) were prepared for each treatment. The test substance was added to 8.4 L of algal nutrient medium augmented with sodium bicarbonate in glass aspirator bottles (capacity 8.7 L). The solutions were mixed for approximately 24 hours using a 9% vortex (of the static liquid depth). The test solutions were removed through the outlet at the bottom of each mixing vessel into 12 replicates of 140 mL in 125 mL Erlenmeyer flasks (no headspace) containing two 14mm glass spheres to facilitate mixing. The test chambers were inoculated with algae (1.0 x 104 cells/mL) and were sealed with ground glass stoppers. Three replicates were sacrificed daily for cell density determination. The test chambers were placed on shaker tables (100 rpm) to keep the algae in suspension. The test was performed under static conditions with no aeration. The algae was cultured in-house from 5 day old stock cultures in log phase growth. Mean test temperature: 24.2°C (sd = 0.1). Continuous light: intensity was 7780 to 8892 Lux. The pH ranged from 7.5 to 7.6 in the test solutions at test initiation and ranged from 8.1 to 9.8 at test termination. Due to the complex nature and limited water solubility of the test substance, the following exceptions to the guideline apply for this study: The concentration of the test substance in solution was not determined prior to use. Test substance analysis was performed on samples of the WAFs at the start of the test (day 0) and at termination (day 4). The initial concentration of the test substance was not maintained at 80% in the three lower loading rates throughout the test (this may be due to biological activity or physical processes in the test chambers). It was appropriate to prepare individual treatment solutions by adding the test substance to dilution water and removing the WAF of each mixture for testing rather than to prepare dilutions of a stock solution. The test duration was 96 hours, instead of 72 hours. However, both 72 and 96-hour endpoints were determined. None of the above exceptions are believed to have affected the outcome, integrity, or quality of the study.

Reference substance (positive control):

not specified

Key result

Duration:

72 h

Dose descriptor:

other: ErL50

Effect conc.:

1.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.94 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.47 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.3 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

other: ErL50

Effect conc.:

1.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

0.47 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

As this study is a WAF procedure for a stream, the actual loading rate will fulfil the endpoint but the values cannot be used to predict a PNEC.

Results with reference substance (positive control):

not reported

Reported statistics and error estimates:

95% confidence intervals

Hours	EL50 mg/L	ErC50 mg/L	EbL50 mg/L	EbC50 mg/L
72	1.3 (<0.17*->6.9*)	0.9.4 (0.14 - >5.7*)	1.6 (<0.17*->6.9*)	1.2 (<0.14*->5.7*)
96	1.4 (<0.26->6.9*)	1.0 (0.29-3.5)	1.6 (<0.17*->6.9*)	1.2(<0.14*->5.7*)

 Values in parentheses ( ) are 95% confidence levels

 * Confidence interval exceeded the highest or lowest loading rate or concentration tested

Hours	Average Specific Growth Rate		Area under the growth curves
	NOELR	NOEC	NOELR	NOEC
72	0.47 mg/L	0.30 mg/L	0.17 mg/L	0.14 mg/L
96	0.47 mg/L	0..30 mg/L	0.17 mg/L	0.14 mg/L

Validity criteria fulfilled:

yes

Conclusions:

The effects on growth rate (r) based upon actual loading rates for Dicyclopentadiene/Codimer Concentrate (DCPD/Codimer Concentrate) are: 72 hr ErL50 = 1.3 mg/L; 96 hr ErL50 = 1.4 mg/L; 72 and 96 hr NOELR = 0.47 mg/L. 72 and 96 hr NOEC 0.30 mg/L.

Executive summary:

This is a GLP compliant guideline study considered adequate for assessment. The effects on growth rate (r) based upon actual loading rates for Dicyclopentadiene/Codimer Concentrate (DCPD/Codimer Concentrate) are: 72 hr ErL50 = 1.3 mg/L; 96 hr ErL50 = 1.4 mg/L; 72- and 96-hr NOELR = 0.47 mg/L, 72- and 96 -hr NOEC 0.30 mg/L. Whilst this study will fulfil the REACH endpoint and is considered suitable for assessment, as it is based upon a WAF procedure it would not be suitable for deriving a PNEC.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 January - 17 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Naphtha (petroleum), light steam-cracked, debenzenized, C8-16-cycloalkadiene conc. (LOA Category L2-DCPD)
CAS: 68478-10-4
CRO ID: MRD-19-988
EC: 270-790-1
No test substance characterization was performed by the testing facility. The sample was fully characterised by the sponsor before submission to the laboratory.

Analytical monitoring:

yes

Details on sampling:

On Day 0 (initiation), samples were collected from three treatment group WAFs (0.12, 0.61 and 3.0 mg/L) and the control WAF. Each sample at test termination was collected from individual replicate test chambers for the 0.12, 0.61 and 3.0 mg/L treatment groups and the control group. Duplicate samples were analyzed from the treatment groups collected; and a single sample was analyzed for the control group. All samples (no headspace) were collected in clear 20 mL LEAP glass vials with PTFE septa screw caps and analyzed on the same day of sampling or refrigerated until analysis. All samples retained were stored under refrigerated conditions.

Vehicle:

no

Details on test solutions:

Individual WAFs for the 0.27, 0.61, 1.4 and 3.0 mg/L treatment groups were prepared for each loading and control by adding the appropriate volume of the test substance using gas tight (glass and stainless steel) syringes to 4.0L of dilution media at the surface of the media in 4.3L glass aspirator bottles with Teflon® screw plugs. For ease of dosing slightly above 1.0 μL, the 0.055 and 0.12 mg/L treatment groups were prepared by adding the test substance to 12 L and 20 L of dilution media at media surface in 13.5 L and 21.5L glass aspirator bottles, respectively. The volume of test substance dispensed into the aspirator bottle was calculated using the nominal test concentration, the quantity of dilution media and the specific density of the test substance. The test substance specific density was 0.956 g/mL referenced in the Safety Data Sheet.

The WAFs were mixed with Teflon®-coated stir bars at a slow stirring rate on magnetic stir plates for 23h at room temperature. The vortex was at 10% of the static liquid depth. The control WAF was prepared in the same manner without test substance addition.

At the end of mixing, the WAFs were allowed to settle for 1h and 25 minutes. After mixing and at settling initiation, all WAFs appeared clear and colorless. At the end of the settling period, approximately 100 mL of the WAF solution was removed and discarded, then solutions were removed from the aspirator bottles through the outlet at the bottom of the vessels and placed into the respective test chambers. All test chambers contained no headspace.

At test initiation, each test chamber was inoculated with an appropriate volume of algal culture to achieve an initial cell density of approximately 10,000 cells/mL.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Test System
Pseudokirchneriella subcapitata. Culture date: 02 January 2020

Supplier
Algae were cultured at the testing facility. Initial strain (1648) was provided by UTEX, The Culture Collection of Algae, The University of Texas at Austin, Austin, Texas 78712 USA. A liquid culture of algae was received by the testing facility on 27 September 2016.

Culture Methods
Algae are cultured in approximately 150 mL of nutrient media (same as dilution media without sodium bicarbonate) prepared with deionized water and reagent grade chemicals. Cell counts are performed weekly to ensure that the cells are in log phase of growth and to verify that the culture is axenic. A new culture is started approximately weekly using inoculum from the previous culture. The cultures of P. subcapitata are held at room temperature and under continuous illumination (4440 - 5920 lux) provided by cool-white fluorescent bulbs.

Cell Density
Initial cell density of algae was approximately 10,000 cells/mL in each replicate. The algae culture was collected from a stock culture in exponential (log phase) growth after an incubation period of 4 days.

Test type:

static

Water media type:

other: Deionised water

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Not reported

Test temperature:

The temperature ranged from 23.2 to 23.5 ºC, as continuously monitored (Watchdog V5 monitoring system) in an environmental chamber

pH:

The measurements of pH for the control and treatment groups ranged from 7.6-8.9 throughout the exposure.

At the beginning of the test (Day 0) samples were collected from the treatment groups and control WAFs and pH was measured. At the end of the test at 72h, pH was measured in a composite sample of test chamber replicates used for determination of cell counts from each treatment group and the control. The pH of the control solution increased by 1.3 units by the end of the test.

Dissolved oxygen:

Not reported

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal: 0 (control), 0.055, 0.12, 0.27, 0.61, 1.4, 3.0 mg/L.

Analytical verification of the test solution concentrations for the control, 0.12, 0.61 and 3.0 mg/L was performed at test initiation and termination using BE-SPME. The initial mean measured BE concentrations were 0 (not detected) 2.45, 11.2 and 61.2 μmol as 2,3-dimethylnaphthalene/mL PDMS for the control (0), 0.12, 0.61 and 3.0 mg/L treatment groups, respectively. At 72h, corresponding mean measured concentrations were 0 (not detected), 1.83, 6.77 and 44.0 μmol as 2,3-dimethylnaphthalene/mL PDMS, respectively. Nominal loading rates were used in statistical endpoint evaluations.

Details on test conditions:

Continuous light was supplied to the exposure area via cool-white fluorescent bulbs with the intensity ranging from 4871 - 5023 lux during the exposure period. Intensity was measured using a LI-COR LI-250A light meter and a LI-210 photometric sensor. Lighting was measured daily in five locations (four corners and center) on the stir plate at approximately mid-depth of the test chambers. All replicate test chambers were mixed continuously with a stir bar on multi-position stir plate.

Test Chamber
Test chambers were 50 mL glass Erlenmeyer flasks with PTFE-lined screw caps to prevent contamination, evaporation and/or volatilization. Each chamber contained approximately 64 mL of test solution (no headspace) and a Teflon®-coated stir bar of 16 mm x 7.9 mm. All test chambers were placed on a multi-position magnetic stir-plate to support algal growth in a no head-space environment.

Test chamber identification
Each replicate test chamber was labeled to show study number, treatment group and replicate number.

Selection
Each replicate was inoculated with algae and placed on a multi-position stir plate for the duration of the study. Test chamber placement on the stir plate was randomly assigned daily using a computer-generated randomization schedule (SAS, 2016). The printout of the randomization schedule has been included in the raw data.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

1.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

1.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.96 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

This study was considered acceptable based on a cell density increase of ≥16 fold within three days and a coefficient of variation for average specific growth that did not exceed 7% (study, 1.1%) in the replicate control cultures. The coefficient of variation for section by section (i.e., day to day) specific growth rates in the control replicates was below the guideline criteria of 35% (study, 10%).

Analytical verification of the test solution concentrations for the control, 0.12, 0.61 and 3.0 mg/L was performed at test initiation and termination using BE-SPME. The initial mean measured BE concentrations were 0 (not detected) 2.45, 11.2 and 61.2 μmol as 2,3-dimethylnaphthalene/mL PDMS for the control (0), 0.12, 0.61 and 3.0 mg/L treatment groups, respectively. At 72h, corresponding mean measured concentrations were 0 (not detected), 1.83, 6.77 and 44.0 μmol as 2,3-dimethylnaphthalene/mL PDMS, respectively. Nominal loading rates were used in statistical endpoint evaluations.

The pH measurements are provided in Table 1. The mean values for cell density for the control and the treatment groups are presented in Table 2. Mean values and percent inhibition for the overall average specific growth rate and yield at 72 hours are presented in Table 3. No observation of test substance insolubility (surface slicks, precipitates, and/or adherence to the test chamber) was noted during cell count determinations.

Acute toxicity results are expressed as percent inhibition of growth derived from the average specific growth rate (r) and yield (y) relative to the control. The 10% and 50% Effect Loading (EL10 and EL50) are the loading rates of the test substance in dilution medium calculated to result in a 10% and 50% reduction in growth in a population of test organisms over a specified exposure period. All 72-h endpoints for this study are presented as follows (Values in parentheses are 95% confidence intervals):

Growth Rate: ErL10: 1.3 (0.93 – 1.7) mg/L, ErL50: 1.6 (0.85 – 2.4) mg/L
Yield: EyL10: 0.96 (0.20 – 1.7) mg/L, EyL50: 1.3 (1.2 – 1.5) mg/L

Time weighted mean adjusted nominal loading rates and endpoint results are currently being adjusted to account for losses. Therefore, endpoint values will have a small variation.

Reported statistics and error estimates:

Calculations
Toxicity results are expressed as percent inhibition of growth derived from either the average specific growth rate (r) or yield (y) relative to the control. Percent inhibition for each respective endpoint was calculated as:
%I= ((Xc-Xt)/(X_c)) x 100

where: % I = percent inhibition
Xc = mean endpoint value for the control group
Xt = mean endpoint value for the treatment replicates

Cell density, yield, growth rates, percent inhibition, standard deviations, coefficients of variation and means were calculated from the raw data by application of various formulas using Microsoft Excel® 2013.

The section by section (i.e., each 24 hour interval) and whole test average specific growth rate for the test validity criteria were determined from the following equation:
μi-j = ((ln Xj - ln Xi)/(tj - ti))

where: μi-j = average specific growth rate from time i to j (1/day)
Xi = biomass at time i
Xj = biomass at time j

Yield was calculated as the biomass (cell density) at the end of the test minus the starting biomass for each single vessel of the treatment and control. For the treatment groups and control, a mean value for yield along with variance estimates was calculated.

The EL10 and EL50 values for average specific growth rate and yield were determined based on the percent inhibition relative to the control values. The EL10 and EL50 values and confidence intervals for average specific growth rate and yield were calculated based on non-linear regression calculations with Gompertz 3P and Gompertz 4P, respectively (Ratkowsky, 1990) using JMP® Version 14 (JMP, 2018).

This study was considered acceptable based on a cell density increase of ≥16 fold within three days and a coefficient of variation for average specific growth that did not exceed 7% (study, 1.1%) in the replicate control cultures. The coefficient of variation for section by section (i.e., day to day) specific growth rates in the control replicates was below the guideline criteria of 35% (study, 10%).

Table 1. pH Measurements

 

Nominal Loading  Rate (mg/L)	hour
	0^1	72^2,3
0 (control)	7.6	8.9
0.055	7.7	8.9
0.12	7.7	8.9
0.27	7.7	8.8
0.61	7.6	8.9
1.4	7.7	8.4
3.0	7.7	7.8

 ¹pH measurements were collected from the individual WAFs.

²All replicate test solutions from each treatment group and control were pooled.

³An increase in pH over the course of the test is expected with the growth of algae in a no head-space environment. Note: the pH of the control did not increase by more than 1.5 units during the test, consistent with guideline recommendation.

Table 2. Mean Cell Density (cells/mL)

 

Nominal Loading Rate (mg/L)	hours
	0	24	48	72
0 (control)	1.00 x 10^4	4.96 x 10^4	3.17 x 10^5	1.52 x 10^6
0.055	1.00 x 10^4	5.04 x 10^4	3.17 x 10^5	1.41 x 10^6
0.12	1.00 x 10^4	4.71 x 10^4	3.19 x 10^5	1.47 x 10^6
0.27	1.00 x 10^4	4.92 x 10^4	3.15 x 10^5	1.43 x 10^6
0.61	1.00 x 10^4	4.17 x 10^4	2.67 x 10^5	1.45 x 10^6
1.4	1.00 x 10^4	1.79 x 10^4	1.58 x 10^5	6.00 x 10^5
3.0	1.00 x 10^4	1.50 x 10^4	1.67 x 10^4	1.67 x 10^4

 

Table 3. Mean Values and Percent Inhibition for Yield and Growth Rate at 72h

 

Nominal Loading Rate (mg/L)	72 hours
	Yield	% inhibition^1	Growth Rate^2	% inhibition^1
Control	1.51 x 10^6	NA	1.67	NA
0.055	1.40 x  10^6	7.2	1.65	1.5
0.12	1.46 x 10^6	3.3	1.66	0.66
0.27	1.42 x 10^6	6.1	1.65	1.3
0.61	1.44 x 10^5	4.4	1.66	0.89
1.4	5.90 x 10^5	61	1.36	18
3.0	6.67 x 10^3	100	0.17	90

NA = Not Applicable.

¹Percent inhibition when compared to the control group.

²Overall average specific growth rate at 72 hours (i.e. 0-72 hours).

 

Validity criteria fulfilled:

yes

Conclusions:

All 72h endpoints for this study are presented as follows (Values in parentheses ( ) are 95% confidence intervals):

Growth Rate: ErL10: 1.3 (0.93 - 1.7) mg/L, ErL50: 1.6 (0.85 - 2.4) mg/L
Yield: EyL10: 0.96 (0.20 - 1.7) mg/L, EyL50: 1.3 (1.2 - 1.5) mg/L

The preferred observational endpoint in this study is algal growth rate inhibition because it is not dependent on the test design, whereas yield or biomass depends on both growth rate of the test species as well as test duration and other elements of test design (ECHA, 2014; OECD, 2011).

Time weighted mean adjusted nominal loading rates and endpoint results are currently being adjusted to account for losses. Therefore, endpoint values will have a small variation.

Executive summary:

This study was conducted for the Sponsor to evaluate the effects of the water accommodated fractions (WAFs) of Naphtha (petroleum), light steam-cracked, debenzenized, C8-16-cycloalkadiene conc (Cas: 68478-10-4) on growth of the alga, Pseudokirchneriella subcapitata, in a 72h static dose-response test. This study was conducted following the OECD 201 guideline (OECD, 2011).

WAFs were prepared for each treatment group by adding the appropriate amount of the test substance using gas tight (glass and stainless steel) syringes to dilution media in glass aspirator bottles with PTFE screw plugs. The WAFs were mixed for 23h at room temperature with Teflon®-coated stir bars on magnetic stir plates. The control WAF was prepared in the same manner without test substance addition. At the end of mixing, the solutions were allowed to settle for 1h and 25 minutes. At the end of the settling period, the WAFs were removed from the mixing vessels through the outlet at the bottom of the vessels and placed into nine replicate chambers per treatment group and control. All test chambers contained no headspace. The WAF loading rates tested were control (0), 0.055, 0.12, 0.27, 0.61, 1.4 and 3.0 mg/L.

Analytical verification of the test solution concentrations at test initiation and termination was performed on the control, 0.12, 0.61 and 3.0 mg/L treatment groups using an automated biomimetic extraction technique employing solid phase micro-extraction (BE-SPME). Nominal loading rates were used in statistical endpoint evaluations.

All 72h endpoints for this study are presented as follows (Values in parentheses ( ) are 95% confidence intervals):

Growth Rate: ErL10: 1.3 (0.93 - 1.7) mg/L, ErL50: 1.6 (0.85 - 2.4) mg/L.

Yield: EyL10: 0.96 (0.20 - 1.7) mg/L, EyL50: 1.3 (1.2 - 1.5) mg/L.

The preferred observational endpoint in this study is algal growth rate inhibition because it is not dependent on the test design, whereas yield or biomass depends on both growth rate of the test species as well as test duration and other elements of test design (ECHA, 2014; OECD, 2011).

Time weighted mean adjusted nominal loading rates and endpoint results are currently being adjusted to account for losses. Therefore, endpoint values will have a small variation.

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12th September 2019 - 20 September 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

yes

Remarks:

Temperature of environmental chamber 23.6 to 24.2°C during in-life, protocol specified temperature was to be maintained from 21 to 24°C. Deviation had no impact on the quality or integrity of the data produced for this study.

GLP compliance:

yes

Specific details on test material used for the study:

Naphtha (petroleum), steam-cracked middle arom.
CAS: 68516-20-1
CRO ID: MRD-19-989
No test substance characterization was performed by the testing facility. The sample was fully characterised by the sponsor before submission to the laboratory.

Analytical monitoring:

yes

Details on sampling:

On Day 0 (initiation), samples were collected from three treatment group WAFs (0.15, 0.96 and 6.0 mg/L) and the control WAF. Each sample at test termination was collected from individual replicate test chambers for the 0.15, 0.96 and 6.0 mg/L treatment groups and the control group. Duplicate samples were analyzed from the treatment groups collected; and a single sample was analyzed for the control group. All samples (no headspace) were collected in clear 20 mL LEAP glass vials with PTFE septa screw caps and analyzed on the same day of sampling. All samples retained were stored under refrigerated conditions.

Vehicle:

no

Details on test solutions:

Individual WAFs for the 0.38, 0.96, 2.4 and 6.0 mg/L treatment groups were prepared for each loading and control by adding the appropriate volume of the test substance using gas tight (glass and stainless steel) syringes to 4.0L of dilution media at the water surface in 4.3L glass aspirator bottles with Teflon® screw plugs. For ease of dosing above 1.0 μL, the 0.061 and 0.15 mg/L treatment groups were prepared by adding the test substance in 12 L and 20 L of dilution media in 13.5 L and 21.5L glass aspirator bottles, respectively. The volume of test substance dispensed into the aspirator bottle was calculated using the nominal test concentration, the quantity of dilution media and the specific density of the test substance. The test substance specific density was 0.905 g/mL referenced in the Safety Data Sheet.

The WAFs were mixed with Teflon-coated stir bars at a slow stirring rate on magnetic stir plates for 24 ± 1h at room temperature. The vortex was at ≤10% of the static liquid depth. The control WAF was prepared in the same manner without test substance addition.

At the end of mixing, the WAFs were allowed to settle for 1h ± 30 min. After mixing and at settling initiation, all WAFs appeared clear and colorless. At the end of the settling period, approximately 100 mL of the WAF solution was removed and discarded, then solutions were removed from the aspirator bottles through the outlet at the bottom of the vessels and placed into the respective test chambers. All test chambers contained no headspace.

At test initiation, each test chamber was inoculated with an appropriate volume of algal culture to achieve an initial cell density of approximately 10,000 cells/mL

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Test System:
Pseudokirchneriella subcapitata. Culture date: 12 September 2019

Supplier:
Algae were cultured at the testing facility. Initial strain (1648) was provided by UTEX, The Culture Collection of Algae, The University of Texas at Austin, Austin, Texas 78712 USA. A liquid culture of algae was received by the testing facility on 27 September 2016.

Initial cell density of algae was approximately 10,000 cells/mL in each replicate. The algae culture was collected from a stock culture in exponential (log phase) growth after an incubation period of 4 days.

Culture Methods:

Algae are cultured in approximately 150 mL of nutrient media (same as dilution media without sodium bicarbonate) prepared with deionized water and reagent grade chemicals. Cell counts are performed weekly to ensure that the cells are in log phase of growth and to verify that the culture is axenic. A new culture is started approximately weekly using inoculum from the previous culture. The cultures of P. subcapitata are held at room temperature and under continuous illumination (4440 - 5920 lux) provided by cool-white fluorescent bulbs.

Cell Density and Age at Test Initiation:

Initial cell density of algae was approximately 10,000 cells/mL in each replicate. The algae culture was collected from a stock culture in exponential (log phase) growth after an incubation period of 4 days

Test type:

static

Water media type:

other: Deionised water

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Not reported

Test temperature:

23.6-24.2°C

pH:

The measurements of pH for the control and treatment groups ranged from 7.5 - 8.8 throughout the exposure.
At the end of the test at 72h, pH was measured in a composite sample of test chamber replicates used for determination of cell counts from each treatment group and the control. The pH of the control solution increased by 1.3 units by the end of the test.

Dissolved oxygen:

Not reported

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

0 (control), 0.15, 0.96, 6.0 mg/L.

Initial mean measured BE concentrations were 0 (not detected), 1.38, 8.88 and 62.7 μmol as 2,3-dimethylnaphthalene/mL PDMS for the control (0), 0.15, 0.96 and 6.0 mg/L treatment groups, respectively. At 72h, corresponding mean measured concentrations were 0 (not detected), 1.31, 6.78 and 48.7 μmol as 2,3-dimethylnaphthalene/mL PDMS, respectively.

Details on test conditions:

Continuous light was supplied to the exposure area via cool-white fluorescent bulbs with the intensity ranging from 4871 - 5023 lux during the exposure period. Intensity was measured using a LI-COR LI-250A light meter and a LI-210 photometric sensor. Lighting was measured daily in five locations (four corners and center) on the stir plate at approximately mid-depth of the test chambers. All replicate test chambers were mixed continuously with a stir bar on multi-position stir plate.

Test Chamber
Test chambers were 50 mL glass Erlenmeyer flasks with PTFE-lined screw caps to prevent contamination, evaporation and/or volatilization. Each chamber contained approximately 64 mL of test solution (no headspace) and a Teflon®-coated stir bar of 16 mm x 7.9 mm. All test chambers were placed on a multi-position magnetic stir-plate to support algal growth in a no head-space environment.

Test Chamber Identification
Each replicate test chamber was labeled to show study number, treatment group and replicate number.

Selection
Each replicate was inoculated with algae and placed on a multi-position stir plate for the duration of the study. Test chamber placement on the stir plate was randomly assigned daily using a computer-generated randomization schedule (SAS, 2016). The printout of the randomization schedule has been included in the raw data.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

1.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

This study was considered acceptable based on a cell density increase of ≥16 fold within three days and a coefficient of variation for average specific growth that did not exceed 7% (study, 0.17%) in the replicate control cultures. The coefficient of variation for section by section (i.e., day to day) specific growth rates in the control replicates was below the guideline criteria of 35% (study, 25%).

Analytical verification of the test solution concentrations for the control, 0.15, 0.96 and 6.0 mg/L was performed at test initiation and termination using BE-SPME. The initial mean measured BE concentrations were 0 (not detected) 1.38, 8.88 and 62.7 μmol as 2,3-dimethylnaphthalene/mL PDMS for the control (0), 0.15, 0.96 and 6.0 mg/L treatment groups, respectively. At 72h, corresponding mean measured concentrations were 0 (not detected), 1.31, 6.78 and 48.7 μmol as 2,3-dimethylnaphthalene/mL PDMS, respectively. Nominal loading rates were used in statistical endpoint evaluations.

The measurements of pH for the control and treatment groups ranged from 7.5 - 8.8 throughout the exposure. The pH measurements are provided in Table 1. The mean values for cell density for the control and the treatment groups are presented in Table 2. Mean values and percent inhibition for the overall average specific growth rate and yield at 72 hours are presented in Table 3. No observation of test substance insolubility (surface slicks, precipitates, and/or adherence to the test chamber) was noted during cell count determinations.

Acute toxicity results are expressed as percent inhibition of growth derived from the average specific growth rate (r) and yield (y) relative to the control. The 10% and 50% Effect Loading (EL10 and EL50) are the loading rates of the test substance in dilution medium calculated to result in a 10% and 50% reduction in growth in a population of test organisms over a specified exposure period. All 72h endpoints for this study (based on nominal loading rates) are presented as follows (95% confidence intervals):

Growth Rate: ErL10: 1.7 (1.3 – 2.1) mg/L, ErL50: 4.6 (4.1 – 5.0) mg/L
Yield: EyL10: 0.20 (0 – 0.41) mg/L, EyL50: 2.0 (1.5 – 2.5)

Reported statistics and error estimates:

Toxicity results are expressed as percent inhibition of growth derived from either the average specific growth rate (r) or yield (y) relative to the control. Percent inhibition for each respective endpoint was calculated as:
%I= ((Xc-Xt)/(X_c)) x 100

where: % I = percent inhibition
Xc = mean endpoint value for the control group
Xt = mean endpoint value for the treatment replicates

Cell density, yield, growth rates, percent inhibition, standard deviations, coefficients of variation and means were calculated from the raw data by application of various formulas using Microsoft Excel® 2013.

The section by section (i.e., each 24 hour interval) and whole test average specific growth rate for the test validity criteria were determined from the following equation:
μi-j = ((ln Xj - ln Xi)/(tj - ti))

where: μi-j = average specific growth rate from time i to j (1/day)
Xi = biomass at time i
Xj = biomass at time j

Yield was calculated as the biomass (cell density) at the end of the test minus the starting biomass for each single vessel of the treatment and control. For the treatment groups and control, a mean value for yield along with variance estimates was calculated.

The EL10 and EL50 values for average specific growth rate and yield were determined based on the percent inhibition relative to the control values. The EL10 and EL50 values and confidence intervals for average specific growth rate and yield were calculated based on non-linear regression calculations with Gompertz 3P and Gompertz 4P, respectively (Ratkowsky, 1990) using JMP® Version 14 (JMP, 2018).

This study was considered acceptable based on a cell density increase of ≥16 fold within three days and a coefficient of variation for average specific growth that did not exceed 7% (study, 0.17%) in the replicate control cultures. The coefficient of variation for section by section (i.e., day to day) specific growth rates in the control replicates was below the guideline criteria of 35% (study, 25%).

Table 1. pH Measurements

Nominal loading rate (mg/L)	hour
	0^1	72^2,3
0 (control)	7.5	8.8
0.061	7.7	8.7
0.15	7.6	8.8
0.38	7.7	8.8
0.96	7.7	8.4
2.4	7.7	8.6
6.0	7.6	7.7

¹pH measurements were collected from the individual WAFs.

²All replicate test solutions from each treatment group and control were pooled.

³An increase in pH over the course of the test is expected with the growth of algae in a no head-space environment. Note: the pH of the control did not increase by more than 1.5 units during the test, consistent with guideline recommendation.

 

 

Table 2. Mean Cell Density (cells/mL)

Nominal Loading Rate (mg/L)	Hours
	0	24	48	72
0 (control)	1.00 x 10^4	5.00 x 10^4	3.19 x 10^5	9.58 x 10^5
0.061	1.00 x 10^4	5.25 x 10^4	3.35 x 10^5	9.50 x 10^5
0.15	1.00 x 10^4	5.00 x 10^4	3.27 x 10^5	9.50 x 10^5
0.38	1.00 x 10^4	3.92 x 10^4	2.29 x 10^5	6.58 x 10^5
0.96	1.00 x 10^4	3.13 x 10^4	1.96 x 10^5	6.25 x 10^5
2.4	1.00 x 10^4	2.54 x 10^4	1.40 x 10^5	5.04 x 10^5
6.0	1.00 x 10^4	1.17 x 10^4	1.21 x 10^4	1.42 x 10^4

 

 Table 3. Mean Values and Percent Inhibition for Yield and Growth Rate at 72h

Nominal Loading Rate (mg/L)
	Yield	% inhibition^1	Growth Rate^2	% inhibition^1
Control	9.48 x 10^5	NA	1.52	NA
0.061	9.40 x 10^5	0.88	1.52	0.19
0.15	9.40 x 10^5	0.88	1.52	0.20
0.38	6.48 x 10^5	32	1.39	8.4
0.96	6.15 x 10^5	35	1.38	9.5
2.4	4.94 x 10^5	48	1.31	14
6.0	4.17 x 10^3	100	0.12	92

NA = Not Applicable.

¹Percent inhibition when compared to the control group.

²Overall average specific growth rate at 72 hours (i.e. 0-72 hours).

Validity criteria fulfilled:

yes

Conclusions:

All 72-h endpoints for this study (based on nominal loading rates) are presented as follows:

Growth Rate: ErL10: 1.7 (1.3 - 2.1) mg/L, ErL50: 4.6 (4.1 - 5.0) mg/L
Yield: EyL10: 0.20 (0 - 0.41) mg/L, EyL50: 2.0 (1.5 - 2.5) mg/L

Executive summary:

This study was conducted for the Sponsor to evaluate the effects of the water accommodated fractions (WAFs) of Naphtha (petroleum), steam-cracked middle arom. on growth of the alga,Pseudokirchneriella subcapitata, in a 72h static dose-response test. This study was conducted following the OECD 201 guideline (OECD, 2011).

WAFs were prepared for each treatment group by adding the appropriate amount of the test substance using gas tight (glass and stainless steel) syringes to dilution media in glass aspirator bottles with PTFE screw plugs. The WAFs were mixed for 24h at room temperature with Teflon®-coated stir bars on magnetic stir plates. The control WAF was prepared in the same manner without test substance addition. At the end of mixing, the solutions were allowed to settle for approximately 1h. At the end of the settling period, the WAFs were removed from the mixing vessels through the outlet at the bottom of the vessels and placed into nine replicate chambers per treatment group and control. All test chambers contained no headspace. The WAF loading rates tested were control (0), 0.061, 0.15, 0.38, 0.96, 2.4 and 6.0 mg/L.

Analytical verification of the test solution concentrations at test initiation and termination was performed on the control, 0.15, 0.96 and 6.0 mg/L treatment groups using automated biomimetic extraction technique employing solid phase micro-extraction (BE-SPME). Nominal loading rates were used in statistical endpoint evaluations.

EL10 and EL50 values were determined for average specific growth rate and yield.

All 72h endpoints for this study (based on nominal loading rates) are presented as follows:

Growth Rate: ErL10: 1.7 (1.3 - 2.1) mg/L, ErL50: 4.6 (4.1 - 5.0) mg/L

Yield: EyL10: 0.20 (0 - 0.41) mg/L, EyL50: 2.0 (1.5 - 2.5) mg/L

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25th June 2019 - 03 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Solvent naphtha (coal)
Cas: 65996-79-4
CRO ID: MRD-19-990
EC: 266-013-0
No test substance characterization was performed by the testing facility. The sample was fully characterised by the sponsor before submission to the laboratory.

Analytical monitoring:

yes

Details on sampling:

On Day 0 (initiation), samples were collected from three treatment group WAFs (0.31, 1.9 and 12 mg/L) and the control WAF. Each sample at test termination was collected from an individual replicate test chamber for the 0.31, 1.9 and 12 mg/L treatment groups and the control group. Duplicate samples were analyzed from the treatment groups collected; and a single sample was analyzed for the control group. All samples (no headspace) were collected in clear 20 mL LEAP glass vials with PTFE septa screw caps and analyzed on the same day of sampling. All samples retained were stored under refrigerated conditions.

Vehicle:

no

Details on test solutions:

Individual WAFs for the 0.31, 0.77, 1.9, 4.8 and 12 mg/L treatment groups were prepared for each loading and control by adding the appropriate volume of the test substance using gas tight (glass and stainless steel) syringes to 4.0L of dilution media in 4.3L glass aspirator bottles. For ease of dosing above 1.0 µL, the 0.12 mg/L treatment group was prepared by adding the test substance in 12 L of dilution media in a 13.5 L glass aspirator bottle. The aspirator bottles were sealed with Teflon® screw plugs. The volume of test substance dispensed into the aspirator bottle was calculated using the nominal test concentration, the quantity of dilution media and the specific density of the test substance. The test substance specific density was 0.95 g/mL referenced in the Safety Data Sheet.

The WAFs were mixed with Teflon®-coated stir bars at a slow stirring rate on magnetic stir plates for 23.75 hours at room temperature. The vortex was at 10% of the static liquid depth. The control WAF was prepared in the same manner without test substance addition.

At the end of mixing, the WAFs were allowed to settle for approximately 1.5 h. After mixing and at settling initiation, all batch solutions appeared clear and colorless with the exception of 12 mg/L which contained visible test substance to the surface of the WAF. At the end of the settling period, approximately 100 mL of the WAF solution was removed and discarded, then solutions were removed from the aspirator bottles through the outlet at the bottom of the vessels and placed into the respective test chambers. All test chambers contained no headspace.

At test initiation, each test chamber was inoculated with an appropriate volume of algal culture to achieve an initial cell density of approximately 10,000 cells/mL.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Test System:
Pseudokirchneriella subcapitata Culture date: 24 June 2019

Supplier:
Algae were cultured at the testing facility. Initial strain (1648) was provided by UTEX, The Culture Collection of Algae, The University of Texas at Austin, Austin, Texas 78712 USA. A liquid culture of algae was received by the testing facility on 27 September 2016.

Initial cell density of algae was approximately 10,000 cells/mL in each replicate. The algae culture was collected from a stock culture in exponential (log phase) growth after an inoculation period of 4 days.

Culture methods:
Algae are cultured in approximately 150 mL nutrient media (same as dilution media without sodium bicarbonate) prepared with deionized water and reagent grade chemicals. Cell counts are performed weekly to ensure that the cells are in log phase of growth and to verify that the culture is axenic. A new culture is started approximately weekly using inoculum from the previous culture. The cultures of P. subcapitata are held at room temperature and under continuous illumination (4440 - 5920 lux) provided by cool-white fluorescent bulbs.

Cell density and age at test initiation:
Initial cell density of algae was approximately 10,000 cells/mL in each replicate. The algae culture was collected from a stock culture in exponential (log phase) growth after an inoculation period of 4 days.

Test type:

static

Water media type:

other: Deionised water

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Not reported

Test temperature:

The temperature ranged from 23.9 to 24.3 ºC, as continuously monitored (Watchdog V5 monitoring system) in the environmental chamber.

pH:

The measurements of pH for the control and treatment groups ranged from 7.6 - 8.9 throughout the exposure.

At the end of the test at 72h, pH was measured in a composite sample of test chamber replicates used for determination of cell counts from each treatment group and the control. The pH of the control solution increased by 1.2 units by the end of the test.

Dissolved oxygen:

Not reported

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 0.12, 0.31, 0.77, 1.9, 4.8, 12 mg/L.
Analytical verification of the test solution concentrations at test initiation and termination was performed on the control, 0.31, 1.9 and 12 mg/L treatment groups using automated biomimetic extraction technique employing solid phase micro-extraction (BE-SPME). Nominal loading rates were used in statistical endpoint evaluations.

Details on test conditions:

Continuous light was supplied to the exposure area via cool-white fluorescent bulbs with the intensity ranging from 4871 - 5023 lux during the exposure period. Intensity was measured using a LI-COR LI-250A light meter and a LI-210 photometric sensor. Lighting was measured daily in five locations (four corners and center) on the stir plate at approximately mid-depth of the test chambers. All replicate test chambers were mixed continuously with a stir bar on multi-position stir plate.

Test Chamber

Test chambers were 50 mL glass Erlenmeyer flasks with PTFE-lined screw caps to prevent contamination, evaporation and/or volatilization. Each chamber contained approximately 64 mL of test solution (no headspace) and a Teflon®-coated stir bar of 16 mm x 7.9 mm. All test chambers were placed on a multi-position magnetic stir-plate to support algal growth in a no head-space environment.

Test Chamber Identification

Each replicate test chamber was labeled to show study number, treatment group and replicate number.

Selection

Each replicate was inoculated with algae and placed on a multi-position stir plate for the duration of the study. Test chamber placement on the stir plate was randomly assigned daily using a computer-generated randomization schedule (SAS, 2016). The printout of the randomization schedule has been included in the raw data.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

6.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

1.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

This study was considered acceptable based on a cell density increase of ≥16 fold within three days and a coefficient of variation for average specific growth that did not exceed 7% (study, 1.0%) in the replicate control cultures. The coefficient of variation for section by section (i.e., day to day) specific growth rates in the control replicates was below the guideline criteria of 35% (study, 13%).

Analytical verification of the test solution concentrations for the control, 0.31, 1.9 and 12 mg/L was performed at test initiation and termination using BE-SPME. The initial mean measured BE concentrations were 0 (not detected), 1.22, 6.55 and 37.6 µmol as 2,3-dimethylnaphthalene/mL PDMS for the control (0), 0.31, 1.9 and 12 mg/L treatment groups, respectively. At 72h, corresponding measured concentrations were 0 (not detected), 1.11, 4.84 and 29.2 µmol as 2,3-dimethylnaphthalene/mL PDMS, respectively. Nominal loading rates were used in statistical endpoint evaluations. The analytical method and results are presented in Appendix B.

The measurements of pH for the control and treatment groups ranged from 7.6 - 8.9 throughout the exposure. The pH measurements are provided in Table 1. The mean values for cell density for the control and the treatment groups are presented in Table 2. Mean values and percent inhibition for the overall average specific growth rate and yield at 72 hours are presented in Table 3. Individual cell density values, average specific growth rates and yield for each 24h time point are presented in Appendix C, D and E, respectively. No observation of test substance insolubility (surface slicks, precipitates, and/or adherence to the test chamber) was noted during cell count determination.

Acute toxicity results are expressed as percent inhibition of growth derived from the average specific growth rate (r) and yield (y) relative to the control. The 10% and 50% Effect Loading (EL10 and EL50) are the loading rates of the test substance in dilution medium calculated to result in a 10% and 50% reduction in growth in a population of test organisms over a specified exposure period.

The 72h endpoints for this study are presented below (Values in parentheses ( ) are 95% confidence intervals):
Growth Rate: ErL10: 1.3 (1.1 – 1.6) mg/L, ErL50: 6.3 (5.7 – 6.9) mg/L
Yield: EyL10: could not calculate, EyL50: 2.0 (1.6 – 2.3)

Reported statistics and error estimates:

Calculations
Toxicity results are expressed as percent inhibition of growth derived from either the average specific growth rate (r) or yield (y) relative to the control. Percent inhibition for each respective endpoint was calculated as:
%I= ((Xc-Xt)/(X_c)) x 100

where: % I = percent inhibition
Xc = mean endpoint value for the control group
Xt = mean endpoint value for the treatment replicates

Cell density, yield, growth rates, percent inhibition, standard deviations, coefficients of variation and means were calculated from the raw data by application of various formulas using Microsoft Excel® 2013.

The section by section (i.e., each 24 hour interval) and whole test average specific growth rate for the test validity criteria were determined from the following equation:
μi-j = ((ln Xj - ln Xi)/(tj - ti))

where: μi-j = average specific growth rate from time i to j (1/day)
Xi = biomass at time i
Xj = biomass at time j

Yield was calculated as the biomass (cell density) at the end of the test minus the starting biomass for each single vessel of the treatment and control. For the treatment groups and control, a mean value for yield along with variance estimates was calculated.

The EL10 and EL50 values for average specific growth rate and yield were determined based on the percent inhibition relative to the control values. The EL10 and EL50 values and confidence intervals for average specific growth rate and yield were calculated based on non-linear regression calculations with Gompertz 3P and Gompertz 4P, respectively (Ratkowsky, 1990) using JMP® Version 14 (JMP, 2018).

This study was considered acceptable based on a cell density increase of ≥16 fold within three days and a coefficient of variation for average specific growth that did not exceed 7% (study, 0.67%) in the replicate control cultures. The coefficient of variation for section by section (i.e., day to day) specific growth rates in the control replicates was below the guideline criteria of 35% (study, 27%).

Table 1. pH Measurements

 

Nominal Loading  Rate (mg/L)	hour
	0^1	72^2,3
0 (control)	7.7	8.9
0.12	7.7	8.9
0.31	7.7	8.8
0.77	7.6	8.9
1.9	7.7	8.4
4.8	7.7	8.1
12	7.6	7.7

NA = Not applicable.

¹pH measurements were collected from the individual WAFs.

²All replicate test solutions from each treatment group and control were pooled.

³Anincrease in pH over the course of the test is expected with the growth of algae in a no head-space environment. Note: the pH of the control did not increase by more than 1.5 units during the test, consistent with guideline recommendation.

Table 2. Mean Cell Density (cells/mL)

 

Nominal Loading Rate (mg/L)	hours
	0	24	48	72
0 (control)	1.00 x 10^4	4.92 x 10^4	2.90 x 10^5	1.14 x 10^5
0.12	1.00 x 10^4	4.96 x 10^4	2.90 x 10^5	1.15 x 10^5
0.31	1.00 x 10^4	5.04 x 10^4	2.88 x 10^5	1.12 x 10^5
0.77	1.00 x 10^4	3.63 x 10^4	1.96 x 10^5	8.33 x 10^5
1.9	1.00 x 10^4	2.63 x 10^4	1.45 x 10^5	5.17 x 10^5
4.8	1.00 x 10^4	1.92 x 10^4	3.46 x 10^4	2.13 x 10^5
12	1.00 x 10^4	1.25 x 10^4	1.25 x 10^4	1.17 x 10^4

 

Table 3. Mean Values and Percent Inhibition for Yield and Growth Rate at 72h

 

Nominal Loading Rate (mg/L)	72 hours
	Yield	% inhibition^1	Growth Rate^2	% inhibition^1
Control	1.13 x 10^6	NA	1.58	NA
0.12	8.19 x 10^6	-0.74	1.58	-0.16
0.31	1.11 x 10^6	2.2	1.57	0.45
0.77	8.23x 10^5	27	1.47	6.7
1.9	5.07 x 10^5	55	1.31	17
4.8	2.03x 10^5	82	1.02	36
12	1.67 x 10^3	100	0.051	97

NA = Not Applicable.

¹Percent inhibition when compared to the control group.

²Overall average specific growth rate at 72 hours (i.e. 0-72 hours).

 

Validity criteria fulfilled:

yes

Conclusions:

The 72h endpoints for this study are presented below (Values in parentheses ( ) are 95% confidence intervals):
Growth Rate: ErL10: 1.3 (1.1 - 1.6) mg/L, ErL50: 6.3 (5.7 - 6.9) mg/L
Yield: EyL50: 2.0 (1.6 - 2.3) mg/L

The preferred observational endpoint in this study is algal growth rate inhibition because it is not dependent on the test design, whereas yield or biomass depends on both growth rate of the test species as well as test duration and other elements of test design (ECHA, 2014; OECD, 2011).

Executive summary:

This study was conducted for theSponsorto evaluate the effects of the water accommodated fractions (WAFs) of Solvent naphtha (coal) (CAS: 65996 -79 -4) on growth of the alga,Pseudokirchneriella subcapitata, in a 72h static dose-response test. This study was conducted following the OECD 201 guideline (OECD, 2011).

 

WAFs were prepared for each treatment group by adding the appropriate amount of the test substance using gas tight (glass and stainless steel) syringes to dilution media in glass aspirator bottles with PTFE screw plugs. The batch solutions were mixed for approximately 24h at room temperature with Teflon^®-coated stir bars on magnetic stir plates. The control batch solution was prepared in the same manner without the test substance addition. At the end of mixing, the solutions were allowed to settle for 1.5h. At the end of the settling period, the solutions were removed from the mixing vessels through the outlet at the bottom of the vessels and placed into nine replicate chambers. All test chambers contained no headspace. The WAF loading rates tested were control (0), 0.12, 0.31, 0.77, 1.9, 4.8 and 12 mg/L.

 

Analytical verification of the test solution concentrations at test initiation and termination was performed on the control, 0.31, 1.9 and 12 mg/L treatment groups using automated biomimetic extraction technique employing solid phase micro-extraction (BE-SPME). Nominal loading rates were used in statistical endpoint evaluations. 

 

EL10 and EL50 values were determined for average specific growth rate and yield.

The 72h endpoints for this study are presented below (Values in parentheses (  ) are 95% confidence intervals):

Growth Rate: ErL10: 1.3 (1.1 - 1.6) mg/L, ErL50: 6.3 (5.7 - 6.9) mg/L

Yield: EyL10: could not calculate, EyL50: 2.0 (1.6 - 2.3) mg/L

The preferred observational endpoint in this study is algal growth rate inhibition because it is not dependent on the test design, whereas yield or biomass depends on both growth rate of the test species as well as test duration and other elements of test design (ECHA, 2014; OECD, 2011).

Reference 6

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 September 2019 - 17 September 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Distillates (petroleum), cracked, ethylene manuf. by-product, C9-10 fraction (LOA Category L2 - DCPD)
CAS: 94733-07-0
EC: 305-586-4
CRO ID:MRD-19-982
No test substance characterization was performed by the testing facility. The sample was fully characterised by the sponsor before submission to the laboratory.

Analytical monitoring:

yes

Details on sampling:

On Day 0 (initiation), samples were collected from three treatment group WAFs (0.14, 0.88 and 5.5 mg/L) and the control WAF. Each sample at test termination was collected from individual replicate test chambers for the 0.14, 0.88 and 5.5 mg/L treatment groups and the control group. Duplicate samples were analyzed from the treatment groups collected; and a single sample was analyzed for the control group. All samples (no headspace) were collected in clear 20 mL LEAP glass vials with PTFE septa screw caps and analyzed on the same day of sampling. All samples retained were stored under refrigerated conditions.

Vehicle:

no

Details on test solutions:

Individual WAFs for the 0.35, 0.88, 2.2 and 5.5 mg/L treatment groups were prepared for each loading and control by adding the appropriate volume of the test substance using gas tight (glass and stainless steel) syringes to 4.0L of dilution media at the water surface in 4.3L glass aspirator bottles with Teflon® screw plugs. For ease of dosing above 1.0 μL, the 0.056 and 0.14 mg/L treatment groups were prepared by adding the test substance in 12 L and 20 L of dilution media in 13.5 L and 21.5L glass aspirator bottles, respectively. The volume of test substance dispensed into the aspirator bottle was calculated using the nominal test concentration, the quantity of dilution media and the specific density of the test substance. The test substance specific density was 0.91 g/mL referenced in the Safety Data Sheet.

The WAFs were mixed with Teflon-coated stir bars at a slow stirring rate on magnetic stir plates for 24 ± 1h at room temperature. The vortex was at ≤10% of the static liquid depth. The control WAF was prepared in the same manner without test substance addition.

At the end of mixing, the WAFs were allowed to settle for 1h ± 30 min. After mixing and at settling initiation, all WAFs appeared clear and colorless. At the end of the settling period, approximately 100 mL of the WAF solution was removed and discarded, then solutions were removed from the aspirator bottles through the outlet at the bottom of the vessels and placed into the respective test chambers. All test chambers contained no headspace.

At test initiation, each test chamber was inoculated with an appropriate volume of algal culture to achieve an initial cell density of approximately 10,000 cells/mL.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Test System
Pseudokirchneriella subcapitata. Culture date: 05 September 2019

Supplier
Algae were cultured at the testing facility. Initial strain (1648) was provided by UTEX, The Culture Collection of Algae, The University of Texas at Austin, Austin, Texas 78712 USA. A liquid culture of algae was received by the testing facility on 27 September 2016.

Initial cell density of algae was approximately 10,000 cells/mL in each replicate. The algae culture was collected from a stock culture in exponential (log phase) growth after an incubation period of 4 days.

Culture Methods

Algae are cultured in approximately 150 mL of nutrient media (same as dilution media without sodium bicarbonate) prepared with deionized water and reagent grade chemicals. Cell counts are performed weekly to ensure that the cells are in log phase of growth and to verify that the culture is axenic. A new culture is started approximately weekly using inoculum from the previous culture. The cultures of P. subcapitata are held at room temperature and under continuous illumination (4440 - 5920 lux) provided by cool-white fluorescent bulbs.

Cell Density and Age at Test Initiation
Initial cell density of algae was approximately 10,000 cells/mL in each replicate. The algae culture was collected from a stock culture in exponential (log phase) growth after an incubation period of 4 days.

Test type:

static

Water media type:

other: Deionised water

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Not reported

Test temperature:

The temperature ranged from 22.9 to 24.3 ºC, as continuously monitored (Watchdog V5 monitoring system) in an environmental chamber

pH:

The measurements of pH for the control and treatment groups ranged from 7.6 - 8.8 throughout the exposure. The pH measurements are provided in Table 1.

The pH of the control solution increased by 1.1 units by the end of the test.

Dissolved oxygen:

Not reported

Salinity:

Not reported

Nominal and measured concentrations:

Nominal: Control (0), 0.14, 0.88 and 5.5 mg/L.

Analytical verification of the test solution concentrations was performed at test initiation and termination using BE-SPME. The initial mean measured BE concentrations were 0 (not detected) 1.16, 7.95 and 52.9 µmol as 2,3-dimethylnaphthalene/mL PDMS for the control (0), 0.14, 0.88 and 5.5 mg/L treatment groups, respectively. At 72h, corresponding mean measured concentrations were 0 (not detected), 1.05, 6.04 and 44.4 µmol as 2,3-dimethylnaphthalene/mL PDMS, respectively. Nominal loading rates were used in statistical endpoint evaluations.

Details on test conditions:

Continuous light was supplied to the exposure area via cool-white fluorescent bulbs with the intensity ranging from 4871 - 5023 lux during the exposure period. Intensity was measured using a LI-COR LI-250A light meter and a LI-210 photometric sensor. Lighting was measured daily in five locations (four corners and center) on the stir plate at approximately mid-depth of the test chambers. All replicate test chambers were mixed continuously with a stir bar on multi-position stir plate.

Test Chamber
Test chambers were 50 mL glass Erlenmeyer flasks with PTFE-lined screw caps to prevent contamination, evaporation and/or volatilization. Each chamber contained approximately 64 mL of test solution (no headspace) and a Teflon®-coated stir bar of 16 mm x 7.9 mm. All test chambers were placed on a multi-position magnetic stir-plate to support algal growth in a no head-space environment.

Test Chamber Identification
Each replicate test chamber was labeled to show study number, treatment group and replicate number.

Selection
Each replicate was inoculated with algae and placed on a multi-position stir plate for the duration of the study. Test chamber placement on the stir plate was randomly assigned daily using a computer-generated randomization schedule (SAS, 2016). The printout of the randomization schedule has been included in the raw data.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

1.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

1.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

This study was considered acceptable based on a cell density increase of ≥16 fold within three days and a coefficient of variation for average specific growth that did not exceed 7% (study, 0.67%) in the replicate control cultures. The coefficient of variation for section by section (i.e., day to day) specific growth rates in the control replicates was below the guideline criteria of 35% (study, 27%).

Analytical verification of the test solution concentrations for the control, 0.14, 0.88 and 5.5 mg/L was performed at test initiation and termination using BE-SPME. The initial mean measured BE concentrations were 0 (not detected) 1.16, 7.95 and 52.9 µmol as 2,3-dimethylnaphthalene/mL PDMS for the control (0), 0.14, 0.88 and 5.5 mg/L treatment groups, respectively. At 72h, corresponding mean measured concentrations were 0 (not detected), 1.05, 6.04 and 44.4 µmol as 2,3-dimethylnaphthalene/mL PDMS, respectively. Nominal loading rates were used in statistical endpoint evaluations. The analytical method and results are presented in Appendix C.

The measurements of pH for the control and treatment groups ranged from 7.6 - 8.8 throughout the exposure. The pH measurements are provided in Table 1. The mean values for cell density for the control and the treatment groups are presented in Table 2. Mean values and percent inhibition for the overall average specific growth rate and yield at 72 hours are presented in Table 3. No observation of test substance insolubility (surface slicks, precipitates, and/or adherence to the test chamber) was noted during cell count determination.

Acute toxicity results are expressed as percent inhibition of growth derived from the average specific growth rate (r) and yield (y) relative to the control. The 10% and 50% Effect Loading (EL10 and EL50) are the loading rates of the test substance in dilution medium calculated to result in a 10% and 50% reduction in growth in a population of test organisms over a specified exposure period. All 72h endpoints for this study are presented in the following table.

Growth Rate: ErL10: 1.4 (1.2 – 1.5) mg/L, ErL50: 4.0 (3.6 – 4.4) mg/L
Yield: EyL10: 0.31 (0 – 2.2) mg/L, EyL50: 1.9 (0 – 4.4)

Reported statistics and error estimates:

Toxicity results are expressed as percent inhibition of growth derived from either the average specific growth rate (r) or yield (y) relative to the control. Percent inhibition for each respective endpoint was calculated as:
%I= ((Xc-Xt)/(X_c)) x 100

where: % I = percent inhibition
Xc = mean endpoint value for the control group
Xt = mean endpoint value for the treatment replicates

Cell density, yield, growth rates, percent inhibition, standard deviations, coefficients of variation and means were calculated from the raw data by application of various formulas using Microsoft Excel® 2013.

The section by section (i.e., each 24 hour interval) and whole test average specific growth rate for the test validity criteria were determined from the following equation:
μi-j = ((ln Xj - ln Xi)/(tj - ti))

where: μi-j = average specific growth rate from time i to j (1/day)
Xi = biomass at time i
Xj = biomass at time j

Yield was calculated as the biomass (cell density) at the end of the test minus the starting biomass for each single vessel of the treatment and control. For the treatment groups and control, a mean value for yield along with variance estimates was calculated.

The EL10 and EL50 values for average specific growth rate and yield were determined based on the percent inhibition relative to the control values. The EL10 and EL50 values and confidence intervals for average specific growth rate and yield were calculated based on non-linear regression calculations with Gompertz 3P and Gompertz 4P, respectively (Ratkowsky, 1990) using JMP® Version 14 (JMP, 2018).

This study was considered acceptable based on a cell density increase of ≥16 fold within three days and a coefficient of variation for average specific growth that did not exceed 7% (study, 0.67%) in the replicate control cultures. The coefficient of variation for section by section (i.e., day to day) specific growth rates in the control replicates was below the guideline criteria of 35% (study, 27%).

Table 1. pH Measurements

 

Nominal Loading  Rate (mg/L)	hour
	0^1	72^2,3
0 (control)	7.7	8.8
0.056	7.6	8.8
0.14	7.6	8.7
0.35	7.7	8.6
0.88	7.6	8.4
2.2	7.7	8.2
5.5	7.6	7.7

NA = Not applicable.

¹pH measurements were collected from the individual WAFs.

²All replicate test solutions from each treatment group and control were pooled.

³An increase in pH over the course of the test is expected with the growth of algae in a no head-space environment. Note: the pH of the control did not increase by more than 1.5 units during the test, consistent with guideline recommendation.

Table 2. Mean Cell Density (cells/mL)

 

Nominal Loading Rate (mg/L)	hours
	0	24	48	72
0 (control)	1.00 x 10^4	4.96 x 10^4	3.00 x 10^5	8.38 x 10^5
0.056	1.00 x 10^4	5.04 x 10^4	2.96 x 10^5	8.29 x 10^5
0.14	1.00 x 10^4	4.71 x 10^4	2.88 x 10^5	8.42 x 10^5
0.35	1.00 x 10^4	4.83 x 10^4	2.85 x 10^5	7.29 x 10^5
0.88	1.00 x 10^4	2.75 x 10^4	1.60 x 10^5	6.13 x 10^5
2.2	1.00 x 10^4	1.38 x 10^4	5.58 x 10^4	3.79 x 10^5
5.5	1.00 x 10^4	1.25 x 10^4	1.25 x 10^4	1.33 x 10^4

 

Table 3. Mean Values and Percent Inhibition for Yield and Growth Rate at 72h

 

Nominal Loading Rate (mg/L)	72 hours
	Yield	% inhibition^1	Growth Rate^2	% inhibition^1
Control	8.28 x 10^5	NA	1.48	NA
0.056	8.19 x 10^5	1.0	1.47	0.24
0.14	8.32 x 10^5	-0.50	1.48	-0.11
0.35	7.19 x 10^5	13	1.43	3.1
0.88	6.03 x 10^5	27	1.37	7.1
2.2	3.69 x 10^5	55	1.21	18
5.5	3.33 x 10^3	100	0.094	94

NA = Not Applicable.

¹Percent inhibition when compared to the control group.

²Overall average specific growth rate at 72 hours (i.e. 0-72 hours).

 

Validity criteria fulfilled:

yes

Conclusions:

All 72h endpoints for this study are presented in the following table.

Growth Rate: ErL10: 1.4 (1.2 - 1.5) mg/L, ErL50: 4.0 (3.6 - 4.4) mg/L
Yield: EyL10: 0.31 (0 - 2.2) mg/L, EyL50: 1.9 (0 - 4.4) mg/L

Executive summary:

This study was conducted for theSponsorto evaluate the effects of the water accommodated fractions (WAFs) of Distillates (petroleum), cracked, ethylene manuf. by-product, C9-10 fraction (LOA Category L2 - DCPD) (CAS: 94733-07-0) on growth of the alga, Pseudokirchneriella subcapitata, in a 72h static dose-response test. This study was conducted following the OECD 201 guideline (OECD, 2011).

 

WAFs were prepared for each treatment group by adding the appropriate amount of the test substance using gas tight (glass and stainless steel) syringes to dilution media in glass aspirator bottles with PTFE screw plugs. The WAFs were mixed for 24h at room temperature with Teflon^®-coated stir bars on magnetic stir plates. The control WAF was prepared in the same manner without test substance addition. At the end of mixing, the solutions were allowed to settle for approximately 1h. At the end of the settling period, the WAFs were removed from the mixing vessels through the outlet at the bottom of the vessels and placed into nine replicate chambers. All test chambers contained no headspace. The WAF loading rates tested werecontrol (0), 0.056, 0.14, 0.35, 0.88, 2.2 and 5.5 mg/L.

 

Analytical verification of the test solution concentrations at test initiation and termination was performed on the control, 0.14, 0.88 and 5.5 mg/L treatment groups using automated biomimetic extraction technique employing solid phase micro-extraction (BE-SPME). Nominal loading rates were used in statistical endpoint evaluations. 

 

EL10 and EL50 values were determined for average specific growth rate and yield. All 72h endpoints for growth rate (r) and yield (y) are presented below.

Growth Rate: ErL10: 1.4 (1.2 - 1.5) mg/L, ErL50: 4.0 (3.6 - 4.4) mg/L

Yield: EyL10: 0.31 (0 - 2.2) mg/L, EyL50: 1.9 (0 - 4.4) mg/L

Reference 7

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

See Attached Justification

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on QSARs R.6

Principles of method if other than guideline:

- Software tool(s) used including version:
See Field 'Attached justification'
- Model(s) used:
See Field 'Attached justification'
- Model description: See Field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'

GLP compliance:

not specified

Specific details on test material used for the study:

HC Block Type Carbon Number Concentration (%w/w)
Mono-Aromatic (MoAr) 7 2.49
8 4.24
9 66.33
10 5.12
Naphthenic Mono-Aromatic (NMAr) 9 19.58
Total 97.76

The constituents that could fit in more than one HC block, or that did not fit in any specific block, were placed in a relevant block that presented the highest toxicity.

Analytical monitoring:

not required

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Test type:

other: QSAR

Total exposure duration:

72 h

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

2.99 mg/L

Nominal / measured:

estimated

Conc. based on:

other: QSAR prediction

Basis for effect:

growth rate

Remarks on result:

other: QSAR predicted value

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.574 mg/L

Nominal / measured:

estimated

Conc. based on:

other: QSAR prediction

Basis for effect:

growth rate

Remarks on result:

other: QSAR predicted value

Validity criteria fulfilled:

yes

Conclusions:

The QSAR predicted 72-h EL50 (growth rate) and 72-h EL10 (growth rate) for P. subcapitata were 2.99 and 0.574 mg/L, respectively.

Executive summary:

The QSAR predicted 72 -h EL50 (growth rate) and 72 -h EL10 (growth rate) of the UVCB substance for P. subcapitata were 2.99 and 0.574 mg/L, respectively. The model used for the calculations was the PETROTOX v4.0 model. PETROTOX is a well-documented and peer reviewed model that is widely used across the chemicals industry. This substance fits within the criteria of the model and there are no reservations about the validity of the model runs. It is expected that this data is reliable with restrictions.

Reference 8

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

See Attached Justification

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on QSARs R.6

Principles of method if other than guideline:

- Software tool(s) used including version:
See Field 'Attached justification'
- Model(s) used:
See Field 'Attached justification'
- Model description: See Field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'

GLP compliance:

not specified

Specific details on test material used for the study:

HC Block Type Carbon Number Concentration (%w/w)
Mono-Aromatic (MoAr) 6 0.44
7 2.99
8 5.10
9 83.11
10 0.95
Naphthenic Mono-Aromatic (NMAr) 9 2.40
Di-Aromatic (DiAr) 10 1.13
n-Olefin 10 0.54
n-Paraffin (n-P) 10 0.14
11 0.33
Total 97.12

The constituents that could fit in more than one HC block, or that did not fit in any specific block, were placed in a relevant block that presented the highest toxicity.

Analytical monitoring:

not required

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Test type:

other: QSAR

Total exposure duration:

72 h

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

2.81 mg/L

Nominal / measured:

estimated

Conc. based on:

other: QSAR prediction

Basis for effect:

growth rate

Remarks on result:

other: QSAR predicted value

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.537 mg/L

Nominal / measured:

estimated

Conc. based on:

other: QSAR prediction

Basis for effect:

growth rate

Remarks on result:

other: QSAR predicted value

Validity criteria fulfilled:

yes

Conclusions:

The QSAR predicted 72-h EL50 (growth rate) and 72-h EL10 (growth rate) for P. subcapitata were 2.81 and 0.537mg/L, respectively.

Executive summary:

The QSAR predicted 72 -h EL50 (growth rate) and 72 -h EL10 (growth rate) of the UVCB substance for P. subcapitata were 2.81 and 0.537 mg/L, respectively. The model used for the calculations was the PETROTOX v4.0 model. PETROTOX is a well-documented and peer reviewed model that is widely used across the chemicals industry. This substance fits within the criteria of the model and there are no reservations about the validity of the model runs. It is expected that this data is reliable with restrictions.

Reference 9

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

See Attached Justification

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on QSARs R.6

Principles of method if other than guideline:

- Software tool(s) used including version:
See Field 'Attached justification'
- Model(s) used:
See Field 'Attached justification'
- Model description: See Field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'

GLP compliance:

not specified

Specific details on test material used for the study:

HC Block Type Carbon Number Concentration (%w/w)
Mono-Aromatic (MoAr) 7 1.55
8 31.91
9 17.86
10 2.89
12 2.29
13 0.67
Di-Aromatic (DiAr) 10 1.03
13 0.28
14 0.24
15 0.28
n-Olefin 5 0.27
10 21.06
11 0.41
i-Olefin 9 0.30
Total 81.03

The constituents that could fit in more than one HC block, or that did not fit in any specific block, were placed in a relevant block that presented the highest toxicity.

Analytical monitoring:

not required

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Test type:

other: QSAR

Total exposure duration:

72 h

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.76 mg/L

Nominal / measured:

estimated

Conc. based on:

other: QSAR prediction

Basis for effect:

growth rate

Remarks on result:

other: QSAR predicted value

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.146 mg/L

Nominal / measured:

estimated

Conc. based on:

other: QSAR prediction

Basis for effect:

growth rate

Remarks on result:

other: QSAR predicted value

Validity criteria fulfilled:

yes

Conclusions:

The QSAR predicted 72-h EL50 (growth rate) and 72-h EL10 (growth rate) for P. subcapitata 0.76 and 0.146 mg/L, respectively.

Executive summary:

The QSAR predicted 72 -h EL50 (growth rate) and 72 -h EL10 (growth rate) of the UVCB substance for P. subcapitata were 0.76 and 0.146 mg/L, respectively. The model used for the calculations was the PETROTOX v4.0 model. PETROTOX is a well-documented and peer reviewed model that is widely used across the chemicals industry. This substance fits within the criteria of the model and there are no reservations about the validity of the model runs. It is expected that this data is reliable with restrictions.

Reference 10

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

See Attached Justification

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on QSARs R.6

Principles of method if other than guideline:

- Software tool(s) used including version:
See Field 'Attached justification'
- Model(s) used:
See Field 'Attached justification'
- Model description: See Field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'

GLP compliance:

not specified

Specific details on test material used for the study:

HC Block Type Carbon Number Concentration (%w/w)
Mono-Aromatic (MoAr) 6 0.57
7 0.19
9 0.50
12 0.98
13 0.31
Di-Aromatic (DiAr) 10 0.27
n-Olefin 5 0.63
10 62.38
11 23.68
Total 89.52

The constituents that could fit in more than one HC block, or that did not fit in any specific block, were placed in a relevant block that presented the highest toxicity.

Analytical monitoring:

not required

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Test type:

other: QSAR

Total exposure duration:

72 h

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.48 mg/L

Nominal / measured:

estimated

Conc. based on:

other: QSAR prediction

Basis for effect:

growth rate

Remarks on result:

other: QSAR predicted value

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.092 mg/L

Nominal / measured:

estimated

Conc. based on:

other: QSAR prediction

Basis for effect:

growth rate

Remarks on result:

other: QSAR predicted value

Validity criteria fulfilled:

yes

Conclusions:

The QSAR predicted 72-h EL50 (growth rate) and 72-h EL10 (growth rate) for P. subcapitata 0.48 and 0.092 mg/L, respectively.

Executive summary:

The QSAR predicted 72 -h EL50 (growth rate) and 72 -h EL10 (growth rate) of the UVCB substance for P. subcapitata were 0.48 and 0.092 mg/L, respectively. The model used for the calculations was the PETROTOX v4.0 model. PETROTOX is a well-documented and peer reviewed model that is widely used across the chemicals industry. This substance fits within the criteria of the model and there are no reservations about the validity of the model runs. It is expected that this data is reliable with restrictions.

Reference 11

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

See Attached Justification

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on QSARs R.6

Principles of method if other than guideline:

- Software tool(s) used including version:
See Field 'Attached justification'
- Model(s) used:
See Field 'Attached justification'
- Model description: See Field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'

GLP compliance:

not specified

Specific details on test material used for the study:

HC Block Type Carbon Number Concentration (%w/w)
Mono-Aromatic (MoAr) 7 0.75
8 35.64
9 17.80
10 2.99
12 0.35
13 0.23
Di-Aromatic (DiAr) 10 0.22
n-Cyclohexane (n-CC6) 7 0.11
iso-Naphthenic (i-N) 8 0.10
n-Olefin 5 0.58
6 0.17
9 0.61
10 23.76
i-Olefin 6 0.21
n-Paraffin (n-P) 7 0.12
8 0.18
9 0.30
Total 84.13

The constituents that could fit in more than one HC block, or that did not fit in any specific block, were placed in a relevant block that presented the highest toxicity.

Analytical monitoring:

not required

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Test type:

other: QSAR

Total exposure duration:

72 h

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

1.09 mg/L

Nominal / measured:

estimated

Conc. based on:

other: QSAR prediction

Basis for effect:

growth rate

Remarks on result:

other: QSAR predicted value

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.208 mg/L

Nominal / measured:

estimated

Conc. based on:

other: QSAR prediction

Basis for effect:

growth rate

Remarks on result:

other: QSAR predicted value

Validity criteria fulfilled:

yes

Conclusions:

The QSAR predicted 72-h EL50 (growth rate) and 72-h EL10 (growth rate) for P. subcapitata were 1.09 and 0.208 mg/L, respectively.

Executive summary:

The QSAR predicted 72 -h EL50 (growth rate) and 72 -h EL10 (growth rate) of the UVCB substance for P. subcapitata were 1.09 and 0.208 mg/L, respectively. The model used for the calculations was the PETROTOX v4.0 model. PETROTOX is a well-documented and peer reviewed model that is widely used across the chemicals industry. This substance fits within the criteria of the model and there are no reservations about the validity of the model runs. It is expected that this data is reliable with restrictions.

Reference 12

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

See Attached Justification

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on QSARs R.6

Principles of method if other than guideline:

- Software tool(s) used including version:
See Field 'Attached justification'
- Model(s) used:
See Field 'Attached justification'
- Model description: See Field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'

GLP compliance:

not specified

Specific details on test material used for the study:

HC Block Type Carbon Number Concentration (%w/w)
n-olefin 5 0.57
9 1.24
10 76.50
11 14.84
12 1.21
Total 94.37

The constituents that could fit in more than one HC block, or that did not fit in any specific block, were placed in a relevant block that presented the highest toxicity.

Analytical monitoring:

not required

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Test type:

other: QSAR

Total exposure duration:

72 h

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.48 mg/L

Nominal / measured:

estimated

Conc. based on:

other: QSAR prediction

Basis for effect:

growth rate

Remarks on result:

other: QSAR predicted value

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.092 mg/L

Nominal / measured:

estimated

Conc. based on:

other: QSAR prediction

Basis for effect:

growth rate

Remarks on result:

other: QSAR predicted value

Validity criteria fulfilled:

yes

Conclusions:

The QSAR predicted 72-h EL50 (growth rate) and 72-h EL10 (growth rate) for P. subcapitata were 0.48 and 0.092 mg/L, respectively.

Executive summary:

The QSAR predicted 72 -h EL50 (growth rate) and 72 -h EL10 (growth rate) of the UVCB substance for P. subcapitata were 0.48 and 0.092 mg/L, respectively. The model used for the calculations was the PETROTOX v4.0 model. PETROTOX is a well-documented and peer reviewed model that is widely used across the chemicals industry. This substance fits within the criteria of the model and there are no reservations about the validity of the model runs. It is expected that this data is reliable with restrictions.

Reference 13

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

See Attached Justification

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on QSARs R.6

Principles of method if other than guideline:

- Software tool(s) used including version:
See Field 'Attached justification'
- Model(s) used:
See Field 'Attached justification'
- Model description: See Field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'

GLP compliance:

not specified

Specific details on test material used for the study:

HC Block Type Carbon Number Concentration (%w/w)
Mono-Aromatic (MoAr) 8 1.51
9 42.57
10 7.04
Naphthenic Mono-Aromatic (NMAr) 9 0.98
10 0.36
Di-Aromatic (DiAr) 10 8.24
11 0.85
n-Olefin 5 0.22
9 0.13
10 26.65
11 4.67
Total 93.21

The constituents that could fit in more than one HC block, or that did not fit in any specific block, were placed in a relevant block that presented the highest toxicity.

Analytical monitoring:

not required

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Test type:

other: QSAR

Total exposure duration:

72 h

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.98 mg/L

Nominal / measured:

estimated

Conc. based on:

other: QSAR prediction

Basis for effect:

growth rate

Remarks on result:

other: QSAR predicted value

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.188 mg/L

Nominal / measured:

estimated

Conc. based on:

other: QSAR prediction

Basis for effect:

growth rate

Remarks on result:

other: QSAR predicted value

Validity criteria fulfilled:

yes

Conclusions:

The QSAR predicted 72-h EL50 (growth rate) and 72-h EL10 (growth rate) for P. subcapitata 0.98 and 0.188 mg/L, respectively.

Executive summary:

The QSAR predicted 72 -h EL50 (growth rate) and 72 -h EL10 (growth rate) of the UVCB substance for P. subcapitata were 0.98 and 0.188 mg/L, respectively. The model used for the calculations was the PETROTOX v4.0 model. PETROTOX is a well-documented and peer reviewed model that is widely used across the chemicals industry. This substance fits within the criteria of the model and there are no reservations about the validity of the model runs. It is expected that this data is reliable with restrictions.

Description of key information

Toxicity to aquatic algae data are available for 5 streams within this Category. The ErL10 72h growth rate ranged 1.3 -1.7 mg/L (WAF) and the ErL50 72h growth rate ranged from 1.3 to 6.3 mg/L. The ErL10 72h yield ranged 0.2 -0.96 mg/L (WAF) and the ErL50 72h yield ranged from 1.3 to 2 mg/L. The 96 hour ErL50 ranged 1.4 - 1.5 mg/L (WAF). On the basis of measured concentrations, the 72 hour NOEC ranged 0.30-0.37 mg/L and the 96 hour NOEC ranged 0.30 - 0.94 mg/L.

 

In addition to the experimental data, the aquatic toxicity was predicted by a QSAR, the PETROTOX v4.0 computer model. PETROTOX is a well-documented and peer reviewed model that is widely used across the chemicals industry. The substances of this category fit within the criteria of the model and there are no reservations about the validity of the model runs. The predicted EL50 values for Pseudokirchneriella subcapitata ranged from 0.48 to 2.99 mg/L. The predicted EL10 values for Pseudokirchneriella subcapitata ranged from 0.092 to 0.574 mg/L.

Additional information

Experimental data:

 

EC 270-790-1 / CAS 68478-10-4 (EMBSI, 2020): This is a GLP compliant, guideline study considered adequate for assessment. The experimental EL50 value for Pseudokirchneriella subcapitata 72h growth rate and yield were repectively 1.6 and 1.3 mg/L. The experimental EL10 value for Pseudokirchneriella subcapitata 72h grown rate and biomass were repectively 1.3 and 0.96 mg/L.

 

EC 271-138-9 / CAS 68516-20-1 (EMBSI, 2020): This is a GLP compliant, guideline study considered adequate for assessment. The experimental EL50 value for Pseudokirchneriella subcapitata 72h growth rate and yield were repectively 4.6 and 2 mg/L. The experimental EL10 value for Pseudokirchneriella subcapitata 72h grown rate and biomass were repectively 1.7 and 0.2 mg/L.

 

EC 266-013-0 / CAS 65996-79-4 (EMBSI, 2019): This is a GLP compliant, guideline study considered adequate for assessment. The experimental EL50 value for Pseudokirchneriella subcapitata 72h growth rate and yield were repectively 1.3 and 2 mg/L. The experimental EL10 value for Pseudokirchneriella subcapitata 72h grown rate was 1.3 mg/L.

 

EC 305-586-4 / CAS 94733-07-0 (EMBSI, 2019): This is a GLP compliant, guideline study considered adequate for assessment. The experimental EL50 value for Pseudokirchneriella subcapitata 72h growth rate and yield were repectively 4 and 1.9 mg/L. The experimental EL10 value for Pseudokirchneriella subcapitata 72h grown rate and biomass were repectively 1.4 and 0.31 mg/L.

 

EC 270-790-1 / CAS 68478-10-4 (ACC, 2004g): This is a GLP compliant guideline study considered adequate for assessment. The effects on growth rate (r) based upon actual loading rates for Dicyclopentadiene/Codimer Concentrate (DCPD/Codimer Concentrate) give a 72 hour ErL50 of 1.3 mg/L (WAF), a 96 hour ErL50 of 1.4 mg/L (WAF) and 72 and 96 hour NOELR of 0.47 mg/L (WAF). On the basis of measured concentrations, the 72 and 96 hour NOEC is 0.30 mg/L.

 

EC 270 -737 -2 / CAS No 68477-54-3 (ACC, 2004h): This is a GLP compliant, guideline study considered adequate for assessment. The effects on growth rate (r) based upon actual loading rates for Low Dicyclopentadiene Resin Oil (Low DCPD Resin Oil) gave a 72 and 96 hour ErL50 of 1.5 mg/L (WAF), a 72 hour NOELR of 0.46 mg/L (WAF) and a 96 hour NOELR of 1.1 mg/L (WAF). On the basis of measured concentrations, the 72 hour NOEC is 0.37 mg/L and the 96 hour NOEC is 0.94 mg/L.

 

QSAR predictions:

 

The PETROTOX v4.0 model combines a partitioning model used to calculate the aqueous concentration of hydrocarbon constituents with the Target Lipid Model used to calculate acute and chronic toxicity of non-polar narcotic chemicals. PETROTOX computes toxicity based on the summation of the aqueous-phase concentrations of hydrocarbon block(s) that represent a hydrocarbon substance and membrane-water partition coefficients (KMW) that describe the partitioning of the hydrocarbons between the water and organism.

 

The predicted values were:

 

Species	Substance CAS No.	EL50 (mg/L)	EL10 (mg/L)
Pseudokirchneriella subcapitata	68516-20-1	1.09	0.208
Pseudokirchneriella subcapitata	101316-62-5	2.99	0.574
Pseudokirchneriella subcapitata	68526-56-7	0.48	0.092
Pseudokirchneriella subcapitata	68477-54-3	0.76	0.146
Pseudokirchneriella subcapitata	94733-07-0	0.98	0.188
Pseudokirchneriella subcapitata	68478-10-4	0.48	0.092
Pseudokirchneriella subcapitata	65996-79-4	2.81	0.537