Reaction product aqueous Phosphoric Acid, 1-Methoxypropan-2-ol and 4,4'-Isopropylidenediphenol-Epichlorohydrin

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product aqueous Phosphoric Acid, 1-Methoxypropan-2-ol and 4,4'-Isopropylidenediphenol-Epichlorohydrin

EC Number:

944-815-8

Molecular formula:

Not possible to assign for a complex UVCB substance

IUPAC Name:

Reaction product aqueous Phosphoric Acid, 1-Methoxypropan-2-ol and 4,4'-Isopropylidenediphenol-Epichlorohydrin

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the following schedule:
Frequency: at 0, 24 and 72 hours
Volume: 3 mL from the appropriate centre of test vessels
Storage: Samples were stored in a freezer (≤-15°C) until analysis.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at the WAF prepared at a loading rate of 100 mg/L but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period. Additionally, reserve samples of 3.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C).

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
The batch of the substance tested was a clear yellow viscous liquid (UVCB) with a purity of 99.2% which was not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item. Preparation of test solutions was performed under dimmed light conditions with glassware wrapped in aluminium foil to minimize exposure to light.

Preparation of test solutions started with loading rates individually prepared at 1.0, 10 and 100 mg/L. A 31-minute period of ultrasonic waves followed by an overnight period of magnetic stirring was applied to ensure maximum dissolution of the test item in test medium. The obtained mixtures were centrifuged at 10000 rpm for 10 minutes. Subsequently, the supernatant was drained from the pelleted undissolved material using a pipette and the obtained Water Accommodated Fractions (WAFs) were used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure.

After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL

Test organisms

Test organisms (species):

other: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) - Unicellular green algae
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In house laboratory culture
- Age of inoculum (at test initiation): Not specified
- Method of cultivation: Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg/L as CaCO3

Test temperature:

Maintained between 23 and 24°C constant to within 1°C.

pH:

Within the limits of 6 to 9 and not varying by more than 1.5 units (test 7.9 to 8.1 and control 8.2 to 8.3)

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal: Control, 1, 10 and 100 mg/L
Measured: 0, 0.74, 8.2 and 48 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass vessels with aluminium caps
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 100 mL vessels filled with 50 mL test or control solutions
- Aeration: No
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 179 x 10^4 cells/mL (mean from all vessels)
- No. of organisms per vessel: Not applicable
- No. of vessels per concentration (replicates): 3 replicates at nominally 1 and 10 mg/L and 6 at nominally 100 mg/L
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes as per OECD guideline (M2 media)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Continuous
- Light intensity and quality: 95 to 97 µE/m2/sec when measured in the photosynthetically effective wavelength of 400 to 700 nm.
- Salinity (for marine algae): Not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions. Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.

- Other: Abnormal appearance of the algae compared to the control was assessed using a microscope at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Combined limit test and range finder
- Justification for using less concentrations than requested by guideline: Combined limit test and range finder
- Range finding study
- Test concentrations: Combined limit test and range finder

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks:

Based on biological relevance

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 48 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks:

Based on biological relevance

Details on results:

Measured test item concentrations:
Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 0.74, 8.2 and 48 mg/L at WAFs prepared at 1.0, 10 and 100 mg/L, respectively. The measured concentrations remained stable throughout the exposure period (102 – 113% relative to initial), except for the WAF prepared at 1.0 mg/L which was measured to be at 122% of initial at the end of the test.

Since no biologically relevant effects (i.e.>10%) were observed at the WAF prepared at 1.0 mg/L of which the actual exposure concentration only slightly exceeded the upper limit of the OECD stability criteria (i.e. 80 -120% of initial), effect parameters were expressed in terms of the initially measured concentrations.

The concentrations measured in the samples taken from solutions with algae were not comparable with the concentrations measured in the samples without algae. However, it can be stated that the presence of the algae had no negative effect on the concentration of the test item in test medium throughout the test. This measured concentration was determined to be the maximum solubility of the potentially soluble constituents of the UVCB substance. As such the EC50 values exceeded the limit of solubility.

Inhibition of growth rate:
Statistically significant growth rate of 9.9% was recorded at the limit concentration after 72 hours of exposure. No biologically relevant growth rate inhibition (i.e. >10%) was measured at any test concentration. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the limit concentration when compared to the control.

Results with reference substance (positive control):

- Results with reference substance valid?
- EC50:
Algae were exposed for a period of 72 hours to K2Cr2O7 (Potassium dichromate) concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L and to a control. The initial cell density was 1.0 x 10^4 cells/mL. The EC50 for growth rate inhibition (72h-ErC50) was 0.90 mg/L with a 95% confidence interval ranging from 0.88 to 0.93 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ErC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

With regards to growth rate, Shapiro-Wilk's test on normal distribution was initially employed followed by Levene's test on variance homogeneity (with residuals). The NOEC was determined by using a two sample t-test procedure.

Any other information on results incl. tables

Growth rate and percentage inhibition for the total test period:

Test item^1 Initially measured conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.729	0.0136	6
0.74	1.714	0.0077	3	0.87
8.2	1.663	0.0432	3	3.8
48	1.558	0.0149	6	9.9^#

1 Test Substance; # effect was statistically significant however biologically not relevant (<10%).

Analytical determinations:

Time of sampling [hours]	Date of sampling	Date of analysis^1	Loading rate^2 [mg/L]	Analyzed concentration [mg/L]	Relative to initial [%]
0	19 Jun 2018	27 Jun 2018	0	n.d.
			1.0	0.740
			10	8.21
			100	48.2
			100^3	49.9
24	20 Jun 2018	27 Jun 2018	0	n.d.	n.a.
			1.0	0.756	102
			10	8.36	102
			100	51.1	106
			100^3,4	60.3	121
72	22 Jun 2018	27 Jun 2018	0	n.d.	n.a.
			1.0	0.900	122
			10	8.34	102
			100	54.4	113
			100^3	44.0	88

1. Samples were stored in the freezer (≤ -15°C) until the day of analysis.

2. Awater accommodated fraction (WAF)prepared at the loading rate.

3. Without algae.
4. The sample was re-diluted as initially applied dilution factor resulted in concentration above the calibration range. Until analysis, 28 Jun 2018, the sample was kept in the auto sampler at room temperature.

n.d. Not detected.

n.a. Not applicable.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, no biologically relevant inhibition of growth rate was recorded at any of the concentrations of the test substance tested.