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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3rd January - 17th January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: US EPA 821-R-02-013

Version / remarks:

USEPA (2002) Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. Fourth Edition. October 2002

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
EC: 270-790-1
CRO ID: MRD-19-988
No test substance characterization was performed by the testing facility. The test item was fully characterised by the sponsor before submission to the laboratory.

Analytical monitoring:

yes

Details on sampling:

On Day 0 (initiation) and Day 1 “old”, samples were collected from four treatment group WAFs (0.04, 0.19, 0.41 and 2.0 mg/L) and the control WAF. Day 5 “new” WAFs and Day 6 “old” were collected from three treatment group (0.04, 0.19, and 0.41 mg/L) and the control WAF. “Old” samples were collected from individual replicate test chambers. Duplicate samples were analyzed from the treatment groups collected; and a single sample was analyzed for the control group.

Vehicle:

no

Details on test solutions:

New WAFs were prepared daily by adding the appropriate volume of test substance using gas tight (glass and stainless steel) syringes to the surface of the dilution water in glass aspirator bottles. The 0.02 mg/L treatment group was prepared in 20L of dilution water in a 21.5L glass aspirator bottle, the 0.04 and 0.09 mg/L treatment groups were prepared in 12L of dilution water in 13.5L glass aspirator bottles, the 0.19 and 0.41 mg/L treatment groups were prepared in 4L of dilution water in 4.3L glass aspirator bottles and the 0.91 and 2.0 mg/L treatment groups and control were prepared in 2L of dilution water in 2.2L glass aspirator bottles. All aspirator bottles were sealed with Teflon® screw caps and placed in an environmental chamber at test temperature with a 16:8 light cycle. The volume of test substance dispensed into each aspirator bottle was calculated using the nominal loading rate, the quantity of dilution water 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 on magnetic stir plates with a ≤10% vortex for 24 ± 1h. At the time of mixing, all batch solutions appeared clear and colorless.

At the end of mixing, the WAFs were allowed to settle for approximately 1h ± 30 mins. After the end of the settling period, approximately 100 mL of solution was removed and discarded, then WAFs 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.

A control was prepared in the same manner without test substance. Daily renewals consisted of fresh control and treatment solutions being distributed into new test vials with the appropriate volume of feed, and the C. dubia parent being transferred from the “old” treatment vials to the “new” treatment vials.

Test organisms (species):

Ceriodaphnia dubia

Details on test organisms:

C. dubia were cultured at the testing facility. The original culture was supplied by Environmental Consulting and Testing Inc. Superior, WI 54880 USA.

A total of 80 female <23-h neonates were used, all released within 4 hour period. Neonates used for this study were from adults that were 6 days old.

C. dubia were maintained in 20 mL glass scintillation vials with approximately 20 mL of reconstituted water (dilution water) supplemented with Na2SeO4 at 2μg/L Se and 1μg/L Vitamin B12 at 25 ± 2°C under a 16 hour light: 8 hour dark photoperiod. Stock cultures were transferred to fresh reconstituted water daily and fed a suspension of Pseudokirchneriella subcapitata and yeast-cereal leaves-trout mixture (YCT). The concentration of P. subcapitata and volume of feeds are documented in the culture log. Algae and YCT are supplied by Environmental Consulting and Testing Inc. Superior, WI 54880 USA.

Stock cultures of test organisms were started at least three weeks before the brood animals were needed. Documentation on survival of brood organisms and the number of offspring is maintained at the testing facility.
Suspension of P. subcapitata (190 μL of a 2.7x10^7 cells/mL) to provide approximately 2.6x10^5 cells/mL and YCT (100 μL) were added to each freshly prepared test chamber daily prior to organism transfer.

Test type:

semi-static

Water media type:

other: Reconstituted Deionised

Limit test:

no

Total exposure duration:

6 d

Post exposure observation period:

None

Hardness:

Reconstituted moderately-hard water (APHA, 2017) was prepared from UV-sterilized, deionized well water and reagent grade salts (NaHCO3, CaSO4, MgSO4, and KCl) and recommended micronutrients Na2SeO4 at 2μg/L Se and 1μg/L Vitamin B12. The hardness of the moderately-hard reconstituted water was 84 mg/L (as CaCO3). Batch water was aerated and covered to protect it from light.
Hardness was measured at beginning of the test in each concentration

Test temperature:

Temperatures in the exposure chambers ranged from 24.7 – 26.0 ºC, and was measured at the beginning and end of each 24h exposure.
Temperature was continuously monitored in the test area using the Watchdog V5 monitoring system. During the in-life phase temperature in the environmental chamber ranged from 24.6 to 25.5°C.

pH:

pH was measured at the beginning and end of each 24h exposure. New solution pH measurements did not vary by more than 0.38 units from the old test solution.
The protocol required that the pH should not vary by more than 0.3 units between corresponding new and old solutions however the pH varied from 0.31-0.38 units on several occasions.
pH day 0 in control was 8.26, and in the treatment groups 8.26-8.43, and day 6 control pH was 7.99 and in treated groups 7.99-8.09.

Dissolved oxygen:

Dissolved oxygen was measured at the beginning and end of each 24h exposure. Dissolved oxygen (DO) remained at or above 6.42 mg/L throughout the test.

Salinity:

Not measured

Conductivity:

Conductivity was measured at beginning of the test in each concentration. In control it was 373.0μs/cm, and in treated groups 361.8-371.3 μs/cm.

Nominal and measured concentrations:

Nominal concentrations were 0 (control), 0.02, 0.04, 0.09, 0.19, 0.41, 0.91, 2.0 mg/L.

Details on test conditions:

Test Chamber / Organism Loading:
Test chambers were 20 mL glass scintillation vials containing one C. dubia and approximately 20 mL of test solution with no headspace. Each chamber was closed with PTFE-lined screw caps to minimize contamination, evaporation and/or volatilization.

Selection:
Test chamber position in the test area and organism assignment followed a randomized block design. The offspring from a single female were distributed evenly among the treatments and the control. Neonates were selected from third broods comprising at least eight neonates from adults that were 6 days old. The study director determined organism suitability. A printout of the randomization schedule generated using JMP v. 14.1 (JMP, 2018) was included in the raw data.

Individual C. dubia were not identified. Each test chamber was labeled with study number, treatment group, replicate, and randomization number.

Discrete Measurements:
Temperature, pH, and dissolved oxygen were measured at the beginning (before renewal) and end of each 24h exposure. Conductivity and hardness were measured at the beginning of the test in each concentration. All fresh test solutions were taken directly from the mixing vessels. Old test solutions from (2) individual replicates, if available, were composited for measurement following organism transfer and neonate counts.

Experimental Evaluation:
Observations for immobilization were performed and recorded daily at 24 ± 1h intervals after in-life initiation. Immobilization was considered the lack of swimming ability within 15 sec after gentle agitation of the test chamber. The adults were transferred via pipette to chambers containing fresh test solution daily. Neonate presence and enumeration from each adult was performed following the adult transfer on a daily basis. After completion of the study, the test organisms were discarded and monitoring of environmental conditions was discontinued.

Exposure Duration:
6 days

Key result

Duration:

6 d

Dose descriptor:

EL10

Effect conc.:

0.85 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

6 d

Dose descriptor:

other: EL20

Effect conc.:

0.89 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

6 d

Dose descriptor:

other: NOEL

Effect conc.:

0.41 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

6 d

Dose descriptor:

other: LOEL

Effect conc.:

0.91 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

This study was considered acceptable as there was no mortality in the control C. dubia for the duration of the study. Additionally, 100% of control C. dubia produced three broods by Day 6 (study termination). The mean number of live offspring produced per adult in the control group surviving at the end of study was 25 neonates. Guideline acceptability criteria states that 80% or greater of control organisms must survive and have an average of 15 or more young per surviving female while 60% of surviving controls must produce three broods.

Analytical verification of the control and test solution concentrations of 0.04, 0.19, 0.41, and 2.0 mg/L (if available), was performed at Day 0 and Day 5 representing “new” solutions and Day 1 and Day 6 representing “old” solutions using BE-SPME by GC-FID and reported as μmol as 2,3-dimethylnaphthalene/mL PDMS. Nominal loading rates were used in statistical endpoint evaluations.

New solution pH measurements did not vary by more than 0.38 units from the old test solution. Dissolved oxygen (DO) remained at or above 6.42 mg/L throughout the test. Temperatures in the exposure chambers ranged from 24.7 - 26.0 ºC.

No observation of test substance insolubility (surface slicks, precipitates, and adherence to the test chamber) was noted during the time of organism observations. No abnormal behavior or appearance was observed in the control or lowest six treatment groups. Lethargic organisms were observed in the 2.0 mg/L treatment group on Day 1. No observations of aborted eggs or immobilized neonates were noted in the control or treatment groups throughout the exposure. Immobilized test organisms were observed in the 2.0 mg/L treatment group, where 50% of organisms were immobilized by Day 1, and 100% of organisms were immobilized by Day 2.

Both acute (LL50) and chronic (EL10, EL20, NOEL, LOEL) endpoints were calculated for this study. The acute endpoint was calculated using 48h immobilization data and the chronic endpoints were calculated using reproduction data gathered throughout the duration of the study.

PROTOCOL DEVIATION

The protocol required that the pH should not vary by more than 0.3 units between corresponding new and old solutions however the pH varied from 0.34-0.57 units on several occasions.

No critical effects were observed in the control group and the validity of the study was maintained.

Results with reference substance (positive control):

No chronic reference toxicity test was performed at the testing facility.

Reported statistics and error estimates:

The EL10 and EL20 chronic endpoints were determined based on neonate counts and nominal loading rates relative to the control value. The EL10 and EL20 values with associated confidence intervals were calculated based on a non-linear regression model, the Hill Model, using the Benchmark Dose method (USEPA, 2010).

The NOEL and LOEL chronic endpoint data was determined based on nominal loading rates and reproduction data using JMP v. 14.1 (JMP, 2018). The Shapiro-Wilk goodness of fit test was used to determine if the data was normally distributed (Shapiro and Wilk, 1965). An ANOVA (Analysis of Variance) was used to determine homogeneity of variance of reproduction data. Nonparametric comparison with control using the Steel method (Steel, 1959) was performed to determine the NOEL and LOEL of nominal loading rates vs. total number of neonates produced in three broods. This statistical analysis identified the 0.02 mg/L level as statistically significant, but due to no effect at the consecutive three higher levels, it may be interpreted that it is not justifiable as a LOEL.

Results table:

Response variable	Endpoints (based on nominal loading rates (mg/L))
	Endpoint	Mean	Confidence interval results
6 day reproduction	EL10	0.85	0.50 -1.20^1
	EL20	0.89	0.70 -1.07^1
	NOEL	0.41
	LOEL	0.91

¹Values are 97.5% confidence intervals

²Values are 99% confidence intervals

 

Validity criteria fulfilled:

yes

Conclusions:

Chronic (EL10, EL20, NOEL, LOEL) endpoints were calculated for this study. The chronic endpoints were calculated using reproduction data gathered throughout the 6 days of the study and were based on nominal rates:
EL10: 0.85 (97.5% confidence interval of 0.50-1.20) mg/L
EL20: 0.89 (97.5% confidence interval of 0.70 -1.07) mg/L
NOEL: 0.41 mg/L
LOEL: 0.91 mg/L

Data above is based on nominal loading rates
*Values are 97.5% confidence intervals
**Values are 99% confidence intervals

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 survival and reproductive output of the cladoceran, Ceriodaphnia dubia, in a 3-brood semi-static dose-response test.

WAFs were prepared daily for each treatment group by adding the appropriate amount of the test substance using gas tight (glass and stainless steel) syringes to moderately hard reconstituted water with micronutrients in glass aspirator bottles sealed with PTFE screw caps. The WAFs were held in an environmental chamber with a 16:8 light cycle at approximately test temperature, and were mixed with Teflon-coated stir bars on magnetic stir plates for approximately 24 ± 1h. 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 1h ± 30 mins. At the end of each settling period, approximately 100 mL of the WAF solution was removed and discarded, then solutions were removed from the mixing vessels through the outlet at the bottom of the vessels and placed into the respective test chambers. All test chambers contained no headspace. The WAF loading rates tested were control (0), 0.02, 0.04, 0.09, 0.19, 0.41, 0.91 and 2.0 mg/L.

Analytical verification of the test solutions for 0.04, 0.19, 0.41 and 2 mg/L and the control were performed on Day 0 and Day 5 “new” solutions and Day 1 and Day 6 “old” solutions, if available, using the automated biomimetic extraction technique employing solid phase micro-extraction (BE-SPME). Nominal loading rates were used in statistical endpoint evaluations.

Chronic (EL10, EL20, NOEL, LOEL) endpoints were calculated for this study. The chronic endpoints were calculated using reproduction data gathered throughout the 6 days of the study and were based on nominal rates:

EL10: 0.85 (97.5% confidence interval of 0.50-1.20) mg/L

EL20: 0.89 (97.5% confidence interval of 0.70 -1.07) mg/L

NOEL: 0.41 mg/L

LOEL: 0.91 mg/L

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 July 2019 - 11 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: US EPA 821-R-02-13

Version / remarks:

USEPA (2002) Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. Fourth Edition. October 2002

Deviations:

yes

Remarks:

No chronic reference toxicity test was performed at the testing facility

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 test item was fully characterised by the sponsor before submission to the laboratory.

Analytical monitoring:

yes

Details on sampling:

On Day 0 (initiation) and Day 5, samples were collected from four treatment group WAFs (0.32, 1.25, 2.5 and 10 mg/L) and the control WAF representing “new” solutions and on Days 1 and 6 representing “old” solutions. Old samples were collected from individual replicates for analysis. 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 the day they were sampled or refrigerated until analysis. All samples retained were stored under refrigerated conditions.

Vehicle:

no

Details on test solutions:

New individual WAFs were prepared daily by adding the appropriate volume of test substance using gas tight (glass and stainless steel) syringes to dilution water in glass aspirator bottles. For the ease of dosing at ~1.0 µl and greater, the 0.16 mg/L treatment group was prepared in 20L of dilution water in a 21.5L glass aspirator bottle, the 0.32, 0.63 and 1.25 mg/L treatment groups were prepared in 12L of dilution water in 13.5L glass aspirator bottles, the 2.5 mg/L treatment group was prepared in 4L of dilution water in 4.3L glass aspirator bottles and the 5.0 and 10.0 mg/L treatment groups and control were prepared in 2L of dilution water in 2.2L glass aspirator bottles. All aspirator bottles were sealed with Teflon® screw caps. The volume of test substance dispensed into each aspirator bottle was calculated using the nominal loading rate, the quantity of dilution water 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 on magnetic stir plates with a ≤10% vortex for an average of 23.4 h at room temperature. At the time of mixing, all batch solutions appeared clear and colorless with the exception of the 10 mg/L treatment which had visible test substance on the surface of the WAF.

At the end of mixing, the WAFs were allowed to settle for approximately 1h. All batch solutions appeared clear and colorless with the exception of the 10 mg/L which contained visible test substance on the surface of the WAF. After the end of the settling period, approximately 100 mL of 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.

A control was prepared in the same manner without test substance. Daily renewals consisted of fresh control and treatment solutions being distributed into new test vials with the appropriate volume of feed, and the C. dubia parent being transferred from the “old” treatment vials to the “new” treatment vials.

Test organisms (species):

Ceriodaphnia dubia

Details on test organisms:

C. dubia were cultured at the testing facility. The original culture was supplied by Environmental Consulting and Testing Inc. Superior, WI 54880 USA.
C. dubia were maintained in 20 mL glass scintillation vials with approximately 20 mL of reconstituted water (dilution water) supplemented with Na2SeO4 at 2µg/L Se and 1µg/L Vitamin B12 at 25 ± 2°C under a 16 hour light: 8 hour dark photoperiod. Stock cultures were transferred to fresh reconstituted water daily and fed a suspension of Pseudokirchneriella subcapitata and yeast-cereal leaves-trout mixture (YCT). The concentration of P. subcapitata and volume of feeds are documented in the culture log. Algae and YCT are supplied by Environmental Consulting and Testing Inc. Superior, WI 54880 USA.

Stock cultures of test organisms were started at least three weeks before the brood animals were needed. Documentation on survival of brood organisms and the number of offspring is maintained at the testing facility
A total of 80 female <24-h neonates were used, all released within ~ 5 h period. The neonates used for this study were from adults that were 6 days old.
Suspension of P. subcapitata (170 µL of a 2.93x107 cells/mL) to provide approximately
2.49x105 cells/mL and YCT (100 µL) were added to each freshly prepared test chamber daily prior to organism transfer.

Test type:

semi-static

Water media type:

other: Reconstituted Deionised

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

None

Hardness:

Reconstituted moderately-hard water (APHA, 2017) was prepared from UV-sterilized, deionized well water and reagent grade salts (NaHCO3, CaSO4, MgSO4, and KCl) and recommended micronutrients Na2SeO4 at 2μg/L Se and 1μg/L Vitamin B12. The hardness of the moderately-hard reconstituted water was 96 mg/L (as CaCO3). Batch water was aerated and covered to protect it from light.
Hardness was measured at beginning of the test in each concentration

Test temperature:

Temperatures in the exposure chambers ranged from 24.0 – 25.5 ºC, and was measured at the beginning and end of each 24h exposure.
Temperature was continuously monitored in the test area using the Watchdog V5 monitoring system. During the in-life phase temperature in the environmental chamber ranged from 24.8 to 25.5 ºC.

pH:

pH was measured at the beginning and end of each 24h exposure. New solution pH measurements did not vary by more than 0.45 units from the old test solution.
The protocol required that the pH should not vary by more than 0.3 units between corresponding new and old solutions however the pH varied from 0.31-0.45 units on several occasions.
pH day 0 in control was 8.11, and in the treatment groups 8.26-8.29, and day 7 control pH was 7.69 and in treated groups 7.84-8.00.

Dissolved oxygen:

Dissolved oxygen was measured at the beginning and end of each 24h exposure. Dissolved oxygen (DO) remained at or above 6.15 mg/L throughout the test.

Salinity:

Not measured

Conductivity:

Conductivity was measured at beginning of the test in each concentration. In control it was 365.0μs/cm, and in treated groups 362.1-369.0μs/cm.

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 0.16, 0.32, 0.63, 1.25, 2.5, 5.0, 10 mg/L.

Details on test conditions:

Test Chamber / Organism Loading:
Test chambers were 20 mL glass scintillation vials containing one C. dubia and approximately 20 mL of test solution with no headspace. Each chamber was closed with PTFE-lined screw caps to minimize contamination, evaporation and/or volatilization.

Selection:
Test chamber position in the test area and organism assignment followed a randomized block design. The offspring from a single female were distributed evenly among the treatments and the control. Neonates were selected from third broods comprising at least eight neonates from adults that were 6 days old. The study director determined organism suitability. A printout of the randomization schedule generated using JMP v. 14.1 (JMP, 2018) was included in the raw data.

Individual C. dubia were not identified. Each test chamber was labeled with study number, treatment group, replicate, and randomization number.

Discrete Measurements:
Temperature, pH, and dissolved oxygen were measured at the beginning (before renewal) and end of each 24h exposure. Conductivity and hardness were measured at the beginning of the test in each concentration. All fresh test solutions were taken directly from the mixing vessels. Old test solutions from (2) individual replicates, if available, were composited for measurement following organism transfer and neonate counts.

Experimental Evaluation:
Observations for immobilization were performed and recorded daily at 24 ± 1h intervals after in-life initiation. Immobilization was considered the lack of swimming ability within 15 sec after gentle agitation of the test chamber. The adults were transferred via pipette to chambers containing fresh test solution daily. Neonate presence and enumeration from each adult was performed following the adult transfer on a daily basis. After completion of the study, the test organisms were discarded and monitoring of environmental conditions was discontinued.

Exposure Duration:
6 days

Key result

Duration:

7 d

Dose descriptor:

EL10

Effect conc.:

0.81 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

This study was considered acceptable as there was no mortality in the control C. dubia for the duration of the study. Additionally, 100% of control C. dubia produced three broods by Day 7 (study termination). The mean number of live offspring produced per adult in the control group surviving at the end of study was 24 neonates. Guideline acceptability criteria states that 80% or greater of control organisms must survive and have an average of 15 or more young per surviving female while 60% of surviving controls must produce three broods.

Analytical verification of the test solution concentrations for the 0.32, 1.25, 2.50 and 10 mg/L was performed at Day 0 and Day 5 representing “new” solutions and Day 1 and Day 6 representing “old” solutions using BE-SPME by GC-FID (µmol as 2,3-dimethylnaphthalene/mL PDMS). The initial (Days 0 and 5 “New”) mean measured concentrations were 0, 1.03, 4.51, 8.23 and 29.3 (Day 0) and 0, 1.66, 4.64 and 8.20 (Day 5) µmol as 2,3-dimethylnaphthalene/mL PDMS for the control (0) 0.32, 1.25, 2.5 and 10 mg/L groups. The old (Days 1 and 6 “old”) mean measured concentrations were 0, 1.21, 3.75, 6.74 and 26.4 (Day 1) and 0, 1.05, 3.93 and 6.82 (Day 6) µmol as 2,3-dimethylnaphthalene/mL PDMS for the control (0) 0.32, 1.25, 2.5 and 10 mg/L groups. Analysis was not conducted at the 10 mg/L loading rate on Days 5 or 6 as all adult Daphnids in this group were immobilized by this point. Nominal loading rates were used in statistical endpoint evaluations.

New solution pH measurements did not vary by more than 0.45 units from the old test solution. Dissolved oxygen (DO) remained at or above 6.15 mg/L throughout the test. Temperatures in the exposure chambers ranged from 24.0 - 25.5 ºC.

No observation of test substance insolubility (surface slicks, precipitates, and adherence to the test chamber) was noted during the time of organism observations. No abnormal behavior or appearance was observed in the control or lowest five treatment groups. No observations of aborted eggs were noted in the control or treatment groups throughout the exposure. Immobilized neonates were observed on Day 5 in 1.25 and 2.5 mg/L treatment groups and on Day 6 in the 1.25 mg/L treatment. There was complete immobilization in the 5.0 mg/L group on Day 2 and 100% immobilization in the 10 mg/L group on Day 1.
Both acute (EL50) and chronic (EL10) endpoints were calculated for this study. The acute endpoint was calculated using 48h immobilization data and the chronic endpoint was calculated using reproduction data gathered throughout the duration of the study.

PROTOCOL DEVIATION

The protocol required that the pH should not vary by more than 0.3 units between corresponding new and old solutions however the pH varied from 0.34-0.57 units on several occasions.

No critical effects were observed in the control group and the validity of the study was maintained.

Results with reference substance (positive control):

No chronic reference toxicity test was performed at the testing facility

Reported statistics and error estimates:

The EL10 chronic endpoint was determined based on percent reproduction inhibition and nominal concentration relative to the control values. The EL10 value with associated confidence intervals was calculated based on a non-linear regression calculation, Logistic 3P (Ratkowsky, 1990).

All acute and chronic endpoints were evaluated using JMP v.14 (JMP, 2018).

Response Variable	Endpoints
	Based on Nominal Loading Rates (mg/L)
Reproduction	EL10	0.81 (0.52–1.09)

Values in parentheses ( ) are 95% confidence intervals.

Validity criteria fulfilled:

yes

Conclusions:

Chronic (EL10) endpoints were calculated for this study. The chronic endpoints were calculated using reproduction data gathered throughout the 6 days of the study and were based on nominal rates:
EL10 = 0.81 (95% confidence interval of 0.52 - 1.09) mg/L.

Executive summary:

This study was conducted for the Sponsor to evaluate the effects of the water accommodated fractions (WAFs) of Solvent naphtha (coal) (CAS: 65996-79-4) on survival and reproductive output of the cladoceran,Ceriodaphnia dubia,in a 3-brood semi-static dose-response test.

 

WAFs were prepared daily for each treatment group by adding the appropriate amount of the test substance using gas tight (glass and stainless steel) syringes to moderately hard reconstituted water with micronutrients in glass aspirator bottles sealed with PTFE screw caps. The batch solutions weremixed for approximately 24h[BCS1] at room temperature with Teflon-coated stir bars on magnetic stir plates. The control batch solution 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 each settling period, approximately100 mL of the WAF solution was removed and discarded, then solutions were removed from the mixing vessels through the outlet at the bottom of the vessels and placed into the respective test chambers. All test chambers contained no headspace. The WAF loading rates tested werecontrol (0), 0.16, 0.32, 0.63, 1.25, 2.5, 5.0 and 10 mg/L.

 

Analytical verification of the test solution concentrations 0.32, 1.25. 5.0 and 10 mg/L and the control were performed on Day 0 and Day 5 “new” solutions and Day 1 and Day 6 “old” solutions using the automated biomimetic extraction technique employing solid phase micro-extraction (BE-SPME). Nominal loading rates were used in statistical endpoint evaluations. 

 

Chronic (EL10) endpoints were calculated for this study. The chronic endpoints were calculated using reproduction data gathered throughout the 6 days of the study and were based on nominal rates:

EL10 = 0.81 (95% confidence interval of 0.52 - 1.09) mg/L.

Reference 3

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5th September 2019 - 17 September 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EPA 821-R-02-13

Version / remarks:

USEPA (2002) Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. Fourth Edition. October 2002

Deviations:

yes

Remarks:

No chronic reference toxicity test was performed at the testing facility

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
CRO ID: MRD-19-982
EC: 305-586-4
No test substance characterization was performed by the testing facility. The test item was fully characterised by the sponsor before submission to the laboratory.

Analytical monitoring:

yes

Details on sampling:

On Day 0 (initiation) and Day 1 “old”, samples were collected from four treatment group WAFs (0.05, 0.32, 0.80 and 5.0 mg/L) and the control WAF. Day 5 “new” WAFs and Day 6 “old” were collected from three treatment groups (0.05, 0.32, and 0.80 mg/L) and the control WAF. “Old” samples were collected from individual replicate test chambers. Duplicate samples were analyzed from the treatment groups collected; and a single sample was analyzed for the control group.

Vehicle:

no

Details on test solutions:

New WAFs were prepared daily by adding the appropriate volume of test substance using gas tight (glass and stainless steel) syringes to the surface of the dilution water in glass aspirator bottles. The 0.02 and the 0.05 mg/L treatment groups were prepared in 20L of dilution water in a 21.5L glass aspirator bottle, the 0.13mg/L treatment group was prepared in 12L of dilution water in 13.5L glass aspirator bottles, the 0.32 and 0.80 mg/L treatment groups were prepared in 4L of dilution water in 4.3L glass aspirator bottles and the 2.0 and 5.0 mg/L treatment groups and control were prepared in 2L of dilution water in 2.2L glass aspirator bottles. All aspirator bottles were sealed with Teflon® screw caps and placed in an environmental chamber at approximately test temperature with a 16:8 light cycle. The volume of test substance dispensed into each aspirator bottle was calculated using the nominal loading rate, the quantity of dilution water and the specific density of the test substance. The test substance specific density was 0.91g/mL referenced in the Safety Data Sheet.
The WAFs were mixed with Teflon® coated stir bars on magnetic stir plates with a ≤10% vortex for an average of 23.5 h. At the time of mixing, all batch solutions appeared clear and colorless with the exception of the 5 mg/L treatment which had visible test substance on the surface of the WAF on Day 0 for approximately 1 h and Days 1 and 2.

At the end of mixing, the WAFs were allowed to settle for approximately 1h. All batch solutions appeared clear and colorless with the exception of the 10 mg/L which contained visible test substance on the surface of the WAF. After the end of the settling period, approximately 100 mL of 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.

Test organisms (species):

Ceriodaphnia dubia

Details on test organisms:

C. dubia were cultured at the testing facility. The original culture was supplied by Environmental Consulting and Testing Inc. Superior, WI 54880 USA.

A total of 80 female <24-h neonates were used, all released within 5 hour period. Neonates used for this study were from adults that were 8 days old.

C. dubia were maintained in 20 mL glass scintillation vials with approximately 20 mL of reconstituted water (dilution water) supplemented with Na2SeO4 at 2μg/L Se and 1μg/L Vitamin B12 at 25 ± 2°C under a 16 hour light: 8 hour dark photoperiod. Stock cultures were transferred to fresh reconstituted water daily and fed a suspension of Pseudokirchneriella subcapitata and yeast-cereal leaves-trout mixture (YCT). The concentration of P. subcapitata and volume of feeds are documented in the culture log. Algae and YCT are supplied by Environmental Consulting and Testing Inc. Superior, WI 54880 USA.

Stock cultures of test organisms were started at least three weeks before the brood animals were needed. Documentation on survival of brood organisms and the number of offspring is maintained at the testing facility.
Suspension of P. subcapitata (190 μL of a 2.71x107 cells/mL) to provide approximately

2.6x105 cells/mL and YCT (100 μL) were added to each freshly prepared test chamber daily prior to organism transfer.

Test type:

semi-static

Water media type:

other: Reconstituted Deionised

Limit test:

no

Total exposure duration:

7 d

Post exposure observation period:

None

Hardness:

Reconstituted moderately-hard water (APHA, 2017) was prepared from UV-sterilized, deionized well water and reagent grade salts (NaHCO3, CaSO4, MgSO4, and KCl) and recommended micronutrients Na2SeO4 at 2μg/L Se and 1μg/L Vitamin B12. The hardness of the moderately-hard reconstituted water was 88 mg/L (as CaCO3). Batch water was aerated and covered to protect it from light.
Hardness was measured at beginning of the test in each concentration

Test temperature:

Temperatures in the exposure chambers ranged from 22.3 – 25.3 ºC, and was measured at teh beginning and end of each 24h exposure.
Temperature was continuously monitored in the test area using the Watchdog V5 monitoring system. During the in-life phase temperature in the environmental chamber ranged from 24.2 to 25.4 ºC.

pH:

pH was measured at the beginning and end of each 24h exposure. New solution pH measurements did not vary by more than 0.53 units from the old test solution.
The protocol required that the pH should not vary by more than 0.3 units between corresponding new and old solutions however the pH varied from 0.31-0.53 units on several occasions.
pH day 0 in control was 8.12, and in the treatment groups 8.27-8.34, and day 7 control pH was 7.66 and in treated groups 7.51-7.88.

Dissolved oxygen:

Dissolved oxygen was measured at the beginning and end of each 24h exposure. Dissolved oxygen (DO) remained at or above 6.25 mg/L throughout the test.

Salinity:

Not measured

Conductivity:

Conductivity was measured at beginning of the test in each concentration. In control it was 385.4μs/cm, and in treated groups 379.6-382.7μs/cm.

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 0.02, 0.05, 0.13, 0.32, 0.80, 2.0, 5.0 mg/L.

Details on test conditions:

Test Chamber / Organism Loading:
Test chambers were 20 mL glass scintillation vials containing one C. dubia and approximately 20 mL of test solution with no headspace. Each chamber was closed with PTFE-lined screw caps to minimize contamination, evaporation and/or volatilization.

Selection:
Test chamber position in the test area and organism assignment followed a randomized block design. The offspring from a single female were distributed evenly among the treatments and the control. Neonates were selected from third broods comprising at least eight neonates from adults that were 6 days old. The study director determined organism suitability. A printout of the randomization schedule generated using JMP v. 14.1 (JMP, 2018) was included in the raw data.

Individual C. dubia were not identified. Each test chamber was labeled with study number, treatment group, replicate, and randomization number.

Discrete Measurements:
Temperature, pH, and dissolved oxygen were measured at the beginning (before renewal) and end of each 24h exposure. Conductivity and hardness were measured at the beginning of the test in each concentration. All fresh test solutions were taken directly from the mixing vessels. Old test solutions from (2) individual replicates, if available, were composited for measurement following organism transfer and neonate counts.

Experimental Evaluation:
Observations for immobilization were performed and recorded daily at 24 ± 1h intervals after in-life initiation. Immobilization was considered the lack of swimming ability within 15 sec after gentle agitation of the test chamber. The adults were transferred via pipette to chambers containing fresh test solution daily. Neonate presence and enumeration from each adult was performed following the adult transfer on a daily basis. After completion of the study, the test organisms were discarded and monitoring of environmental conditions was discontinued.

Exposure Duration:
6 days

Key result

Duration:

7 d

Dose descriptor:

EL10

Effect conc.:

0.57 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

This study was considered acceptable as there was no mortality in the control C. dubia for the duration of the study. Additionally, 100% of control C. dubia produced three broods by Day 7 (study termination). The mean number of live offspring produced per adult in the control group surviving at the end of study was 29 neonates. Guideline acceptability criteria states that 80% or greater of control organisms must survive and have an average of 15 or more young per surviving female while 60% of surviving controls must produce three broods.

Analytical verification of the test solution concentrations for the 0.05, 0.32, 0.80 and 5.0 mg/L was performed at Day 0 and Day 5 representing “new” solutions and Day 1 and Day 6 representing “old” solutions using BE-SPME by GC-FID and reported as µmol as 2,3-dimethylnaphthalene/mL PDMS. Nominal loading rates were used in statistical endpoint evaluations.

New solution pH measurements did not vary by more than 0.53 units from the old test solution. Dissolved oxygen (DO) remained at or above 6.25 mg/L throughout the test. Temperatures in the exposure chambers ranged from 22.3 - 25.3 ºC.

No observation of test substance insolubility (surface slicks, precipitates, and adherence to the test chamber) was noted during the time of organism observations. No abnormal behavior or appearance was observed in the control or lowest five treatment groups. No observations of aborted eggs or immobilized neonates were noted in the control or treatment groups throughout the exposure. Immobilized ceriodaphnids were observed on Day 1 in 2.0 and 5.0 mg/L treatment groups. There was 20% immobilization in the 2.0 mg/L group by Day 4 and 100% immobilization in the 5 mg/L group on Day 2.

Chronic (EL10) endpoints were calculated for this study. The chronic endpoint was calculated using reproduction data gathered throughout the duration of the study

PROTOCOL DEVIATION

The protocol required that the pH should not vary by more than 0.3 units between corresponding new and old solutions however the pH varied from 0.34-0.57 units on several occasions.

No critical effects were observed in the control group and the validity of the study was maintained.

Results with reference substance (positive control):

No chronic reference toxicity test was performed at the testing facility.

Reported statistics and error estimates:

The EL10 chronic endpoint was determined based on percent reproduction inhibition and nominal concentration relative to the control values. The EL10 value with associated confidence intervals was calculated based on a non-linear regression calculation, Logistic 4P (Ratkowsky, 1990).

All acute and chronic endpoints were evaluated using JMP v.14 (JMP, 2018).

Results table:

Response Variable	Endpoints
	Based on Nominal Loading Rates (mg/L)
Reproduction	EL10	0.57 (0.42-0.72)

Values in parentheses ( ) are 95% confidence intervals.

Validity criteria fulfilled:

yes

Conclusions:

Chronic (EL10) endpoints were calculated for this study.The chronic endpoints were calculated using reproduction data gathered throughout the 6 days of the study and were based on nominal rates:
EL10: 0.57 (95% confidence interval of 0.42-0.72) mg/L.

Executive summary:

This study was conducted for the Sponsor to evaluate the effects of the water accommodated fractions (WAFs) of Distillates (petroleum), cracked, ethylene manuf. by-product, C9-10 fraction (CAS: 94733-07-0) on survival and reproductive output of the cladoceran, Ceriodaphnia dubia, in a 3-brood semi-static dose-response test.

 

WAFs were prepared daily for each treatment group by adding the appropriate amount of the test substance using gas tight (glass and stainless steel) syringes to moderately hard reconstituted water with micronutrients in glass aspirator bottles sealed with PTFE screw caps. The WAFs were mixed in an environmental chamber at approximately at test temperature and 16:8 light cycle for 23.5h 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 each settling period, approximately100 mL of the WAF solution was removed and discarded, then solutions were removed from the mixing vessels through the outlet at the bottom of the vessels and placed into the respective test chambers. All test chambers contained no headspace. The WAF loading rates tested werecontrol (0), 0.02, 0.05, 0.13, 0.32, 0.80, 2.0, and 5 mg/L.

 

Analytical verification of the test solution concentrations 0.05, 0.32. 0.80 and 5 mg/L and the control were performed on Day 0 and Day 5 “new” solutions and Day 1 and Day 6 “old” solutions using the automated biomimetic extraction technique employing solid phase micro-extraction (BE-SPME). Nominal loading rates were used in statistical endpoint evaluations. 

 

Chronic (EL10) endpoints were calculated for this study.The chronic endpoints were calculated using reproduction data gathered throughout the 6 days of the study and were based on nominal rates:

EL10: 0.57 (95% confidence interval of 0.42-0.72) mg/L.

Reference 4

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30th September 2019 - 10th October 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: US EPA 821-R-02-013

Version / remarks:

USEPA (2002) Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. Fourth Edition. October 2002

Deviations:

yes

Remarks:

No chronic reference toxicity test was performed at the testing facility

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
EC: 271-138-9
No test substance characterization was performed by the testing facility. The test item was fully characterised by the sponsor before submission to the laboratory.

Analytical monitoring:

yes

Details on sampling:

On Day 0 (initiation) and Day 1 “old”, samples were collected from four treatment group WAFs (0.05, 0.32, 0.80 and 5.0 mg/L) and the control WAF. Day 5 “new” WAFs and Day 6 “old” were collected from three treatment group (0.05, 0.32, and 0.80 mg/L) and the control WAF. “Old” samples were collected from individual replicate test chambers. Duplicate samples were analyzed from the treatment groups collected; and a single sample was analyzed for the control group.

Vehicle:

no

Details on test solutions:

New WAFs were prepared daily by adding the appropriate volume of test substance using gas tight (glass and stainless steel) syringes to the surface of the dilution water in glass aspirator bottles. The 0.02 and the 0.05 mg/L treatment groups were prepared in 20L of dilution water in 21.5L glass aspirator bottles, the 0.13 mg/L treatment group was prepared in 12L of dilution water in a 13.5L glass aspirator bottle, the 0.32 and 0.80 mg/L treatment groups were prepared in 4L of dilution water in 4.3L glass aspirator bottles and the 2.0 and 5.0 mg/L treatment groups and control were prepared in 2L of dilution water in 2.2L glass aspirator bottles. All aspirator bottles were sealed with Teflon® screw caps and placed in an environmental chamber at test temperature with a 16:8 light cycle. The volume of test substance dispensed into each aspirator bottle was calculated using the nominal loading rate, the quantity of dilution water 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 on magnetic stir plates with a ≤10% vortex for 24 ± 1h. At the time of mixing, all batch solutions appeared clear and colorless.
At the end of mixing, the WAFs were allowed to settle for approximately 1h ± 30 mins. After the end of the settling period, approximately 100 mL of solution was removed and discarded, then WAFs 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.
A control was prepared in the same manner without test substance. Daily renewals consisted of fresh control and treatment solutions being distributed into new test vials with the appropriate volume of feed, and the C. dubia parent being transferred from the “old” treatment vials to the “new” treatment vials.
Individual C. dubia were not identified. Each test chamber was labeled with study number, treatment group, replicate, and randomization number.

Test organisms (species):

Ceriodaphnia dubia

Details on test organisms:

C. dubia were cultured at the testing facility. The original culture was supplied by Environmental Consulting and Testing Inc. Superior, WI 54880 USA.

A total of 80 female <24-h neonates were used, all released within 5 hour period. Neonates used for this study were from adults that were 8 days old.

C. dubia were maintained in 20 mL glass scintillation vials with approximately 20 mL of reconstituted water (dilution water) supplemented with Na2SeO4 at 2μg/L Se and 1μg/L Vitamin B12 at 25 ± 2°C under a 16 hour light: 8 hour dark photoperiod. Stock cultures were transferred to fresh reconstituted water daily and fed a suspension of Pseudokirchneriella subcapitata and yeast-cereal leaves-trout mixture (YCT). The concentration of P. subcapitata and volume of feeds are documented in the culture log. Algae and YCT are supplied by Environmental Consulting and Testing Inc. Superior, WI 54880 USA.

Stock cultures of test organisms were started at least three weeks before the brood animals were needed. Documentation on survival of brood organisms and the number of offspring is maintained at the testing facility.
Suspension of P. subcapitata (190 μL of a 2.71x107 cells/mL) to provide approximately 2.6x105 cells/mL and YCT (100 μL) were added to each freshly prepared test chamber daily prior to organism transfer.

Test type:

semi-static

Water media type:

other: Reconstituted Deionised

Limit test:

no

Total exposure duration:

6 d

Post exposure observation period:

None

Hardness:

Reconstituted moderately-hard water (APHA, 2017) was prepared from UV-sterilized, deionized well water and reagent grade salts (NaHCO3, CaSO4, MgSO4, and KCl) and recommended micronutrients Na2SeO4 at 2μg/L Se and 1μg/L Vitamin B12. The hardness of the moderately-hard reconstituted water was 80 mg/L (as CaCO3). Batch water was aerated and covered to protect it from light.

Hardness was measured at beginning of the test in each concentration

Test temperature:

Temperatures in the exposure chambers ranged from 24.0 – 26.0 ºC, and was measured at the beginning and end of each 24h exposure.
Temperature was continuously monitored in the test area using the Watchdog V5 monitoring system. During the in-life phase temperature in the environmental chamber ranged from 25.1 to 25.7 ºC.

pH:

pH was measured at the beginning and end of each 24h exposure. New solution pH measurements did not vary by more than 0.57 units from the old test solution.

The protocol required that the pH should not vary by more than 0.3 units between corresponding new and old solutions however the pH varied from 0.34-0.57 units on several occasions.

pH day 0 in control was 8.34, and in the treatment groups 8.33-8.41, and day 6 control pH was 7.87 and in treated groups 7.81-8.05.

Dissolved oxygen:

Dissolved oxygen was measured at the beginning and end of each 24h exposure. Dissolved oxygen (DO) remained at or above 6.50 mg/L throughout the test.

Salinity:

Not measured

Conductivity:

Conductivity was measured at beginning of the test in each concentration. In control it was 347.1μs/cm, and in treated groups 343.8-347.9μs/cm.

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 0.05, 0.32, 0.8, and 5.0 mg/L.

Details on test conditions:

Test Chamber / Organism Loading:
Test chambers were 20 mL glass scintillation vials containing one C. dubia and approximately 20 mL of test solution with no headspace. Each chamber was closed with PTFE-lined screw caps to minimize contamination, evaporation and/or volatilization.

Selection:
Test chamber position in the test area and organism assignment followed a randomized block design. The offspring from a single female were distributed evenly among the treatments and the control. Neonates were selected from third broods comprising at least eight neonates from adults that were 6 days old. The study director determined organism suitability. A printout of the randomization schedule generated using JMP v. 14.1 (JMP, 2018) was included in the raw data.

Individual C. dubia were not identified. Each test chamber was labeled with study number, treatment group, replicate, and randomization number.

Discrete Measurements:
Temperature, pH, and dissolved oxygen were measured at the beginning (before renewal) and end of each 24h exposure. Conductivity and hardness were measured at the beginning of the test in each concentration. All fresh test solutions were taken directly from the mixing vessels. Old test solutions from (2) individual replicates, if available, were composited for measurement following organism transfer and neonate counts.

Experimental Evaluation:
Observations for immobilization were performed and recorded daily at 24 ± 1h intervals after in-life initiation. Immobilization was considered the lack of swimming ability within 15 sec after gentle agitation of the test chamber. The adults were transferred via pipette to chambers containing fresh test solution daily. Neonate presence and enumeration from each adult was performed following the adult transfer on a daily basis. After completion of the study, the test organisms were discarded and monitoring of environmental conditions was discontinued.

Exposure Duration:
6 days

Key result

Duration:

6 d

Dose descriptor:

EL10

Effect conc.:

0.49 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

6 d

Dose descriptor:

other: EL20

Effect conc.:

0.72 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

6 d

Dose descriptor:

other: NOEL

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

6 d

Dose descriptor:

other: LOEL

Effect conc.:

0.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

This study was considered acceptable as there was no mortality in the control C. dubia for the duration of the study. Additionally, 100% of control C. dubia produced three broods by Day 6 (study termination). The mean number of live offspring produced per adult in the control group surviving at the end of study was 27 neonates. Guideline acceptability criteria states that 80% or greater of control organisms must survive and have an average of 15 or more young per surviving female while 60% of surviving controls must produce three broods.

Analytical verification of the test solution concentrations for the 0.05, 0.32, 0.80 and 5.0 mg/L was performed at Day 0 and Day 5 representing “new” solutions and Day 1 and Day 6 representing “old” solutions using BE-SPME by GC-FID and reported as (μmol as 2,3-dimethylnaphthalene/mL PDMS). Nominal loading rates were used in statistical endpoint evaluations.

New solution pH measurements did not vary by more than 0.57 units from the old test solution. Dissolved oxygen (DO) remained at or above 6.50 mg/L throughout the test. Temperatures in the exposure chambers ranged from 24.0 - 26.0 ºC.

No observation of test substance insolubility (surface slicks, precipitates, and adherence to the test chamber) was noted during the time of organism observations. No abnormal behavior or appearance was observed in the control or lowest five treatment groups. No observations of aborted eggs or immobilized neonates were noted in the control or treatment groups throughout the exposure. Immobilized test organisms were observed on Day 1 in 0.80, 2.0 and 5.0 mg/L treatment groups. There was 10% immobilization in the 0.80 mg/L group by Day 1, 20% immobilization in the 2.0 mg/L group by Day 2 and 100% immobilization in the 5mg/L group by Day 3.

Both acute (LL50) and chronic (EL10, EL20, NOEL, LOEL) endpoints were calculated for this study. The acute endpoint was calculated using 48h immobilization data and the chronic endpoints were calculated using reproduction data gathered throughout the duration of the study.

PROTOCOL DEVIATION

The protocol required that the pH should not vary by more than 0.3 units between corresponding
new and old solutions however the pH varied from 0.34-0.57 units on several occasions.

No critical effects were observed in the control group and the validity of the study was maintained.

Results with reference substance (positive control):

No chronic reference toxicity test was performed at the testing facility.

Reported statistics and error estimates:

The EL10 and EL20 chronic endpoints were determined based on neonate counts and nominal loading rates relative to the control value. The EL10 and EL20 values with associated confidence intervals were calculated based on a non-linear regression model, the Hill Model, using the Benchmark Dose method (USEPA, 2010).

The NOEL and LOEL chronic endpoint data was determined based on nominal loading rates and reproduction data using JMP v. 14.1 (JMP, 2018). The Shapiro-Wilk goodness of fit test was used to determine if the data was normally distributed (Shapiro and Wilk, 1965). An ANOVA (Analysis of Variance) was used to determine homogeneity of variance of reproduction data. Nonparametric comparison with control using the Steel method (Steel, 1959) was performed to determine the NOEL and LOEL of nominal loading rates vs. total number of neonates produced in three broods. This statistical analysis identified the 0.02 mg/L level as statistically significant, but due to no effect at the consecutive three higher levels, it may be interpreted that it is not justifiable as a LOEL.

Results table

Response variable	Endpoints (based on nominal loading rates (mg/L))
	Endpoint	Mean	Confidence interval results
6 day reproduction	EL10	0.49	0.28-0.71^2
	EL20	0.72	0.49-0.94^2
	NOEL	0.32
	LOEL	0.80

¹Values are 95% confidence intervals

²Values are 97.5% confidence intervals

 

Validity criteria fulfilled:

yes

Conclusions:

Chronic (EL10, EL20, NOEL, LOEL) endpoints were calculated for this study. The chronic endpoints were calculated using reproduction data gathered throughout the 6 days of the study and were based on nominal rates:
EL10: 0.49 (97.5% confidence interval of 0.28-0.71) mg/L
EL20: 0.72 (97.5% confidence interval of 0.49 -0.94) mg/L
NOEL: 0.32 mg/L
LOEL: 0.80 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 (CAS: 68477-54-3)on survival and reproductive output of the cladoceran, Ceriodaphnia dubia, in a 3-brood semi-static dose-response test.

WAFs were prepared daily for each treatment group by adding the appropriate amount of the test substance using gas tight (glass and stainless steel) syringes to moderately hard reconstituted water with micronutrients in glass aspirator bottles sealed with PTFE screw caps. The WAFs were held in an environmental chamber with a 16:8 light cycle at approximately test temperature, and were mixed with Teflon-coated stir bars on magnetic stir plates for approximately 24 ± 1h. 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 1h ± 30 mins. At the end of each settling period, approximately 100 mL of the WAF solution was removed and discarded, then solutions were removed from the mixing vessels through the outlet at the bottom of the vessels and placed into the respective test chambers. All test chambers contained no headspace. The WAF loading rates tested were control (0), 0.02, 0.05, 0.13, 0.32, 0.80, 2.0, and 5 mg/L.

Analytical verification of the test solutions 0.05, 0.32. 0.80 and 5 mg/L and the control were performed on Day 0 and Day 5 “new” solutions and Day 1 and Day 6 “old” solutions using the automated biomimetic extraction technique employing solid phase micro-extraction (BE-SPME). Nominal loading rates were used in statistical endpoint evaluations.

Chronic (EL10, EL20, NOEL, LOEL) endpoints were calculated for this study. The chronic endpoints were calculated using reproduction data gathered throughout the 6 days of the study and were based on nominal rates:

EL10: 0.49 (97.5% confidence interval of 0.28-0.71) mg/L

EL20: 0.72 (97.5% confidence interval of 0.49 -0.94) mg/L

NOEL: 0.32 mg/L

LOEL: 0.80 mg/L

Reference 5

Endpoint:

long-term toxicity to aquatic invertebrates

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):

Daphnia magna

Test type:

other: QSAR Calculation

Total exposure duration:

21 d

Reference substance (positive control):

not required

Key result

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

0.493 mg/L

Nominal / measured:

estimated

Conc. based on:

other: QSAR prediction

Basis for effect:

reproduction

Remarks on result:

other: QSAR predicted value

Validity criteria fulfilled:

yes

Conclusions:

The QSAR predicted 21-d EL10 (reproduction) for D. magna was 0.493 mg/L.

Executive summary:

The QSAR predicted 21 -d EL10 (reproduction) of the UVCB substance for D. magna was 0.493 mg/L. 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 6

Endpoint:

long-term toxicity to aquatic invertebrates

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):

Daphnia magna

Test type:

other: QSAR Calculation

Total exposure duration:

21 d

Reference substance (positive control):

not required

Key result

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

1.36 mg/L

Nominal / measured:

estimated

Conc. based on:

other: QSAR prediction

Basis for effect:

reproduction

Remarks on result:

other: QSAR predicted value

Validity criteria fulfilled:

yes

Conclusions:

The QSAR predicted 21-d EL10 (reproduction) for D. magna was 1.36 mg/L.

Executive summary:

The QSAR predicted 21 -d EL10 (reproduction) of the UVCB substance for D. magna was 1.36 mg/L. 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 7

Endpoint:

long-term toxicity to aquatic invertebrates

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):

Daphnia magna

Test type:

other: QSAR Calculation

Total exposure duration:

21 d

Reference substance (positive control):

not required

Key result

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

0.219 mg/L

Nominal / measured:

estimated

Conc. based on:

other: QSAR prediction

Basis for effect:

reproduction

Remarks on result:

other: QSAR predicted value

Validity criteria fulfilled:

yes

Conclusions:

The QSAR predicted 21-d EL10 (reproduction) for D. magna was 0.219 mg/L.

Executive summary:

The QSAR predicted 21 -d EL10 (reproduction) of the UVCB substance for D. magna was 0.219 mg/L. 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:

long-term toxicity to aquatic invertebrates

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):

Daphnia magna

Test type:

other: QSAR Calculation

Total exposure duration:

21 d

Reference substance (positive control):

not required

Key result

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

0.347 mg/L

Nominal / measured:

estimated

Conc. based on:

other: QSAR prediction

Basis for effect:

reproduction

Remarks on result:

other: QSAR predicted value

Validity criteria fulfilled:

yes

Conclusions:

The QSAR predicted 21-d EL10 (reproduction) for D. magna was 0.347 mg/L.

Executive summary:

The QSAR predicted 21 -d EL10 (reproduction) of the UVCB substance for D. magna was 0.347 mg/L. 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:

long-term toxicity to aquatic invertebrates

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):

Daphnia magna

Test type:

other: QSAR Calculation

Total exposure duration:

21 d

Reference substance (positive control):

not required

Key result

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

0.447 mg/L

Nominal / measured:

estimated

Conc. based on:

other: QSAR prediction

Basis for effect:

reproduction

Remarks on result:

other: QSAR predicted value

Validity criteria fulfilled:

yes

Conclusions:

The QSAR predicted 21-d EL10 (reproduction) for D. magna was 0.447 mg/L.

Executive summary:

The QSAR predicted 21 -d EL10 (reproduction) of the UVCB substance for D. magna was 0.447 mg/L. 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:

long-term toxicity to aquatic invertebrates

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):

Daphnia magna

Test type:

other: QSAR Calculation

Total exposure duration:

21 d

Reference substance (positive control):

not required

Key result

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

0.218 mg/L

Nominal / measured:

estimated

Conc. based on:

other: QSAR prediction

Basis for effect:

reproduction

Remarks on result:

other: QSAR predicted value

Validity criteria fulfilled:

yes

Conclusions:

The QSAR predicted 21-d EL10 (reproduction) for D. magna was 0.218 mg/L.

Executive summary:

The QSAR predicted 21 -d EL10 (reproduction) of the UVCB substance for D. magna was 0.218 mg/L. 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:

long-term toxicity to aquatic invertebrates

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):

Daphnia magna

Test type:

other: QSAR Calculation

Total exposure duration:

21 d

Reference substance (positive control):

not required

Key result

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

1.27 mg/L

Nominal / measured:

estimated

Conc. based on:

other: QSAR prediction

Basis for effect:

reproduction

Remarks on result:

other: QSAR predicted value

Validity criteria fulfilled:

yes

Conclusions:

The QSAR predicted 21-d EL10 (reproduction) for D. magna was 1.27 mg/L.

Executive summary:

The QSAR predicted 21 -d EL10 (reproduction) of the UVCB substance for Daphnia magna was  0.1.27 mg/L. 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

There are chronic daphnia studies available for 4 streams in this category. These were all performed in accordance with US EPA 821-R-02-13 using Ceriodaphnia dubia and exposures via WAFs. These studies reported an EL10 for reproduction ranging from 0.49 to 0.85 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 EL10 values for Daphnia magna ranged from 0.218 to 1.27 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 EL10 value for Ceriodaphnia dubia 6 day reproduction was 0.85 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 EL10 value for Ceriodaphnia dubia 6 day reproduction was 0.49 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 EL10 value for Ceriodaphnia dubia 7 day reproduction was 0.81 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 EL10 value for Ceriodaphnia dubia 7 day reproduction was 0.57 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.	EL10 (mg/L)
Daphnia magna	68516-20-1	0.493
Daphnia magna	101316-62-5	1.36
Daphnia magna	68526-56-7	0.219
Daphnia magna	68477-54-3	0.347
Daphnia magna	94733-07-0	0.447
Daphnia magna	68478-10-4	0.218
Daphnia magna	65996-79-4	1.27