Reaction mass of sodium hydrogen N-(1-oxooctadecyl)-L-glutamate and sodium hydrogen N-(1-oxohexadecyl)-L-glutamate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 August 2008 to 18 September 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with GLP and agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Signature: 15/10/2007; Date of Inspection: 21/08/2007

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.17mg/L and 1.7mg/L
- Sampling method: Not stated.
- Sample storage conditions before analysis: Samples were analysed immediately after sampling. Duplicate samples were taken at 0 and 72 hours and stored at approximately -20 °C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test materials, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of the test material in cases where the test material is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (100 mg/l) of test material in culture medium for a period of 24 hours prior to removing any undissolved test material present by filtration (0.2 µm Sartorius Sartopore, first approximate 2 litres discarded in order to pre-condition the filter) to give a saturated solution of the test material.

- Eluate:
Test water.

- Differential loading:
Not applicable.

- Controls:
Test medium control used in each test.

- Positive Control:
A positive control (Safepharm Laboratories Project Number: 0039/0994) used potassium dichromate as the reference material. An amount of reference material (100 mg) was dissolved in culture medium and the volume adjusted to 1 litre to give a 100 mg/l stock solution from which a series of dilutions were made to give further stock solutions of 10, 2.0, 1.0, 0.50, 0.25 and 0.125 mg/l. An aliquot (250 ml) of each of the 0.125, 0.25, 0.50, 1.0 and 2.0 mg/l stock solutions was separately mixed with algal suspension (250 ml) to give the required test concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.
The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Six replicate flasks were prepared for the control and three replicate flasks prepared for each test concentration.
The flasks were incubated (INFORS MultitronâVersion 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter®Multisizer Particle Counter.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
Not applicable.

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
Not applicable.

- Evidence of undissolved material (e.g. precipitate, surface film, etc):
None stated.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: algae
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture collection of Algae and Protozoa (CCAP) Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): Not stated.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 24 ± 1 °C.

ACCLIMATION
- Acclimation period: Not stated.
- Culturing media and conditions (same as test or not): Same as test.
- Any deformed or abnormal cells observed: None stated.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C. Temperature was recorded daily.

pH:

Measured using a WTW pH 320 pH meter.

Control: 0 hours, 7.2-7.3; 72 hours, 7.9-8.1
0.54 mg/L: 0 hours, 7.2; 72 hours, 8.1-8.2
1.3 mg/L: 0 hours, 7.1; 72 hours, 8.1-8.2
4.0 mg/L: 0 hours, 7.1-7.2; 72 hours, 8.1
14 mg/L: 0 hours, 7.2; 72 hours, 8.1
42 mg/L: 0 hours, 7.4; 72 hours, 8.0

Nominal and measured concentrations:

Range finding test:
Nominal: 0.0051, 0.051, 0.51, 5.1 and 51 mg/L
Measured: Not measured.

Definitive test:
Nominal: 0.51, 1.61, 5.1, 16.1 and 51 mg/L
Measured: 0.54, 1.3, 4.0, 14 and 42 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL glass conical flasks containing 100mL of control or test medium.
- Aeration: None.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): Not applicable.
- Initial cells density: initial nominal cell density of 4 x 10^3 cells per mL
- Control end cells density: Mean cell density of control at 72 hours: 4.57 x 10^5 cells per mL
- No. of organisms per vessel: approximately 10^4-10^5 cells/mL
- No. of vessels per concentration (replicates): Range finding test: 2 replicates. Definitive test: 3 replicates
- No. of vessels per control (replicates): Range finding test: 2 replicates. Definitive test: 6 replicates
- No. of vessels per vehicle control (replicates): Not applicable.

GROWTH MEDIUM
- Standard medium used:
yes

- Detailed composition if non-standard medium was used:
NaNO3 25.5 mg/L
MgCl2.6H2O 12.164 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.7 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.1855 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.159 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
Na2SeO3.5H2O 0.000010 mg/L
The culture medium was prepared using reverse osmosis purified deionised water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
* Elga Optima 15+ or Elga Purelab Option R-15 BP

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis, Elga Optima 15+ or Elga Purelab Option R-15 BP
- Culture medium different from test medium: No.
- Intervals of water quality measurement: 72 hours.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: None during test.
- Photoperiod: Continuous illumunation.
- Light intensity and quality: Warm white lighting (380-730nm), approximately 7000 lux.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Samples were taken at 0 and 24 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: For the range test X10
- Range finding study
- Test concentrations: 0.0051, 0.051, 0.51, 5.1 and 51 mg/L.
- Results used to determine the conditions for the definitive study: Based on the results of the range-finding test, test material solutions for the definitive test were prepared by stirring an excess (100 mg/L) of test material in culture medium for a period of time and then removing any undissolved test material by filtration. The saturated solution was then further diluted, as necessary, to produce the remaining test groups.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.51 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: CL = 1.5-2.2 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: CL = 1.6-2.0 mg/L

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.92 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Unusual cell shape: All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures at 72 hours.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The results obtained from the pre-study media preparation trial conducted indicated that a dissolved test material concentrations of 54 and 51 mg/L were obtained following filtration with either the first 1 litre or 2 litres being discarded respectively.
Therefore for the purposes of testing it was considered appropriate to prepare the test material using the saturated solution method of preparation with any undissolved test material removed by filtration through a 0.2 µm Sartorius Sartopore filter pre-conditioned with the first approximate 2 litres.
- Results: The cell densities and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test material during the range-finding test are given in Table 1.
The results showed no significant effect on growth rate at the test concentrations of 0.0051, 0.051 and 0.51 mg/L. However, growth was observed to be reduced at 5.1 and 51 mg/L.
Based on this information the test material solutions for the definitive test were prepared by stirring an excess (100 mg/L) of test material in culture medium for a period of time and then removing any undissolved test material by filtration. This saturated solution was then further diluted, as necessary, to produce the remaining test groups.
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rates, yield and biomass integral values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.
The mean cell densities versus time for the definitive test are presented in Figure 1. Percentage inhibition values are plotted against test concentration in Figure 2, Figure 3, Figure 4, Figure 5. Figure 6 and Figure 7.

Results with reference substance (positive control):

- Results with reference substance valid? Yes

- EC50:
ErC50 (0 - 72 h): 0.57 mg/L; 95% confidence limits 0.48 - 0.66 mg/L
EyC50 (0 - 72 h): 0.32 mg/L; 95% confidence limits 0.29 - 0.35 mg/L
EbC50 (0 - 72 h): 0.31 mg/L; 95% confidence limits 0.28 - 0.35 mg/L

- NOEC:
No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.0625 mg/L
No Observed Effect Concentration (NOEC) based on biomass integral: 0.0625 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.125 mg/L
Lowest Observed Effect Concentration (LOEC) based on biomass integral: 0.125 mg/L

- Other:
The cell densities from exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference material during the positive control are given in Table 5 and Figure 8. Daily specific growth rates for the control cultures are given in Table 6 whilst growth rate, yield and biomass integral values are given in Table 7. Percentage inhibition values are plotted against test concentration in Figure 9, Figure 10 and Figure 11.
The results from the positive control with potassium dichromate were within the normal range for this reference material.

Reported statistics and error estimates:

Statistical analysis
One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate, yield and biomass integral data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 107 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours: 4.29 x 10³cells per mL
Mean cell density of control at 72 hours: 4.57 x 10⁵cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 14 % and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35 %.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 2 % and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7 %.

Growth data

From the data given in Tables 2 and 4, it is clear that the growth rate (r), yield (y) and biomass (b) of Desmodesmus subspicatus (CCAP 276/20) were affected by the presence of the test material over the 72-Hour exposure period.

Accordingly the following results based on the 0-Hour measured test concentrations were determined from the data:

Inhibition of growth rate

E_rC₁₀ (0 - 72 h): 12 mg/L
E_rC₂₀ (0 - 72 h): 18 mg/L
E_rC₅₀ (0 - 72 h): 34 mg/L*

where E_rC_x is the test concentration that reduced growth rate by x %.

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control, 0.54 and 1.3 mg/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 1.3 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 4.0 mg/L.

Inhibition of yield

E_yC₁₀ (0 - 72 h): 3.9 mg/L
E_yC₂₀ (0 - 72 h): 6.2 mg/L
E_yC₅₀ (0 - 72 h): 14 mg/L; 95% confidence limits 11 - 18 mg/L

where E_yC_x is the test concentration that reduced yield by x %.

There were no statistically significant decreases in yield between the control, 0.54 and 1.3 mg/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 1.3 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 4.0 mg/L.

Inhibition of biomass integral

E_bC₁₀ (0 - 72 h): 2.6 mg/L
E_bC₂₀ (0 - 72 h): 4.7 mg/L
E_bC₅₀ (0 - 72 h): 13 mg/L; 95% confidence limits 11 - 16 mg/L

where E_bC_x is the test concentration that reduced biomass integral (area under the growth curve) by x %.

There were no statistically significant decreases in biomass integral between the control, 0.54 and 1.3 mg/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on biomass integral was 1.3 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on biomass integral was 4.0 mg/L.

Observationson cultures

All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures at 72 hours.

Observations on test material solubility

At the start of the test all control, 0.54, 1.3 and 4.0 mg/L test cultures were observed to be clear colourless solutions whilst the 14 and 42 mg/L test cultures were observed to be clear colourless solutions with a slight foam at the media surface. After the 72-Hour test period all control, 0.54, 1.3 and 4.0 mg/L test cultures were observed to be green dispersions. The 14 mg/L test cultures were observed to be pale green dispersions whilst the 42 mg/L test cultures were observed to be slightly hazy very pale green dispersions.

Physico-chemical measurements

The pH values of each test and control flask are given in Table 2. Temperature was maintained at 24 ± 1 ºC throughout the test.

The pH values of the control cultures (see Table 2) were observed to increase from pH 7.2 – 7.3 at 0 hours to pH 7.9 – 8.1 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Verification of test concentrations

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.54 to 42 mg/L. A decline in measured test concentrations in the range of less than the limit of quantitation (LOQ) of the analytical method employed which was determined to be 0.41 mg/L to 0.66 mg/L was observed at 72 hours. This decline was in line with the preliminary stability analyses conducted which indicated that the test material was unstable in culture medium over the test duration.

Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC₅₀ values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.  In cases where the measured concentration was less than the LOQ of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.21 mg/L) was used to enable calculation of the geometric mean measured concentration. The geometric mean measured test concentrations were determined to be:

0-Hour Measured Test Concentration (mg/L)	Geometric Mean Measured Test Concentration (mg/L)	Expressed as a % of the 0- Hour Measured Test Concentration
0.54	0.34	63
1.3	0.51	39
4.0	0.92	23
14	1.7	12
42	5.3	13

The following results were determined from the data based on the geometric mean measured test concentrations:

Growth rate

E_rC₁₀ (0 - 72 h): 1.5 mg/L
E_rC₂₀ (0 - 72 h): 2.2 mg/L
E_rC₅₀ (0 - 72 h): 4.3 mg/L*
No Observed Effect Concentration (NOEC) = 0.51 mg/L
Lowest Observed Effect Concentration (LOEC) = 0.92 mg/L

Yield

E_yC₁₀ (0 - 72 h): 0.79 mg/L
E_yC₂₀ (0 - 72 h): 1.1 mg/L
E_yC₅₀ (0 - 72 h): 1.8 mg/L; 95% confidence limits 1.5 – 2.2 mg/L
No Observed Effect Concentration (NOEC) = 0.51 mg/L
Lowest Observed Effect Concentration (LOEC) = 0.92 mg/L

Biomass integral

E_bC₁₀ (0 - 72 h): 0.66 mg/L
E_bC₂₀ (0 - 72 h): 0.95 mg/L
E_bC₅₀ (0 - 72 h): 1.8 mg/L; 95% confidence limits 1.6 – 2.0 mg/L
No Observed Effect Concentration (NOEC) = 0.51 mg/L
Lowest Observed Effect Concentration (LOEC) = 0.92 mg/L

The use of the geometric mean measured test concentrations in the calculation of the EC₅₀ and NOEC values had a significant effect on the outcome of the study as there was a greater than 10 fold decrease in the EC₅₀ values obtained.

*It was not possible to calculate 95 % confidence limits for the E_rC₅₀ value as the data generated did not fit the models available for the calculation of confidence limits.

Validity criteria fulfilled:

yes

Conclusions:

Based on the geometric mean measured test concentrations the ErC50 (0 - 72 h) value was 4.3 mg/L, the EyC50 (0 - 72 h) value was 1.8 mg/L; 95% confidence limits 1.5 – 2.2 mg/L and the EbC50 (0 - 72 h) value was 1.8 mg/L; 95% confidence limits 1.6 – 2.0 mg/L. The Lowest Observed Effect Concentration based on growth rate, yield and biomass integral was 0.92 mg/L, and the No Observed Effect Concentration was 0.51 mg/L.

Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Whilst the test material was observed to be readily soluble in water, upon addition to culture medium the test material was observed to form a precipitate. A pre-study media preparation trial indicated that a dissolved test material concentration of approximately 51 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to solutions of the test material at 0-Hour measured concentrations of 0.54, 1.3, 4.0, 14 and 42 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test material solutions were prepared by stirring an excess (100 mg/L) of test material in culture medium using a propeller stirrer at approximately 1500 rpm at a temperature of approximately 21°C for 24 hours. After the stirring period any undissolved test material was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a saturated solution of the test material with a 0-Hour measured concentration of 42 mg/L. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using either a Coulter^® Multisizer Particle Counter or a haemocytometer and light microscope.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Desmodesmus subspicatus was exposed to an aqueous solution of the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^® Multisizer Particle Counter.

In terms of growth rate, exposure of Desmodesmus subspicatus to the test material gave an E_rC₅₀(0 - 72 h) value of 34 mg/L*. The Lowest Observed Effect Concentration based on inhibition of growth rate was 4.0 mg/L and the No Observed Effect Concentration was 1.3 mg/L.

In terms of yield, exposure of Desmodesmus subspicatus to the test material gave an E_yC₅₀ (0 - 72 h) value of 14 mg/L; 95% confidence limits 11 - 18 mg/L. The Lowest Observed Effect Concentration based on yield was 4.0 mg/L and the No Observed Effect Concentration was 1.3 mg/L.

In terms of biomass integral (area under growth curve), exposure of Desmodesmus subspicatus to the test material gave an E_bC₅₀(0 - 72 h) value of 13 mg/L; 95% confidence limits 11 - 16 mg/L. The Lowest Observed Effect Concentration based on inhibition of biomass integral was 4.0 mg/L and the No Observed Effect Concentration was 1.3 mg/L.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.54 to 42 mg/L. A decline in measured test concentrations in the range of less than the limit of quantitation (LOQ) of the analytical method employed which was determined to be 0.41 mg/L to 0.66 mg/L was observed at 72 hours. This decline was inline with the preliminary stability analyses conducted which indicated that the test material was unstable in culture medium over the test duration.

Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The E_rC₅₀(0 - 72 h) based on the geometric mean measured test concentrations was 4.3 mg/L*, the E_yC₅₀(0 - 72 h) was 1.8 mg/L; 95% confidence limits 1.5 – 2.2 mg/L, and the    E_bC₅₀(0 - 72 h) was 1.8 mg/L; 95% confidence limits 1.6 – 2.0 mg/L. The Lowest Observed Effect Concentration based on inhibition of growth rate, yield and biomass integral was 0.92 mg/L and the No Observed Effect Concentration was 0.51 mg/L.

Exposure of Desmodesmus subspicatus to the reference material, potassium dichromate, gave an E_rC₅₀(0 - 72 h) of 0.57 mg/L; 95% confidence limits 0.48 – 0.66 mg/L, an E_yC₅₀(0 - 72 h) of 0.32 mg/L; 95% confidence limits 0.29 ‑ 0.35 mg/L, and an E_bC₅₀(0 - 72 h) of 0.31 mg/L; 95% confidence limits 0.28 - 0.35 mg/L. The Lowest Observed Effect Concentrations based on inhibition of growth rate, yield and biomass integral were 0.50, 0.125 and 0.125 mg/L respectively and the No Observed Effect Concentrations were 0.25, 0.0625 and 0.0625 mg/L respectively. 

 

*It was not possible to calculate 95% confidence limits for the E_rC₅₀value as the data generated did not fit the models available for the calculation of confidence limits.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

08 March 2018 to 11 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidelines for studies on the new chemical substance as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law)

Version / remarks:

1973, amended 2009 under the reference of YAKUSHOKHATSU No. 1121002, SEIKYOKU No.2 and KANPOKIHATSU No. 021121002 and partially amended 2006 as the joint ordinance of The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE).

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 2-7-7.The guidelines related to the study reports for the registration application of pesticide

Version / remarks:

Ref. No. 12-Nousan-8147 on 24 November 2000) & Ref. No.13-Seisan-3986 on 10 October 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Analytical measurement was performed at the control and at the applied test concentration levels and from the control at the beginning of the test and in 24 hour intervals thereafter during the experiment.
- Determination of test substance concentrations was performed at each test concentration level and from the additional samples without algae in order to distinguish degradation and adsorption of the test item to algae.
- The samples were analysed by an HPLC-MS method.

Vehicle:

no

Details on test solutions:

- Because the test material is poorly soluble in water, a test material stock solution was prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.
- A saturated test material solution (nominal loading rate of 100.0 mg/L) was prepared by dispersing/dissolving the amount of test material into the test medium (OECD Medium) two days before the start of the study. This solution was shaken for about 24 hours at approximately 30 °C and then equilibrated for about 24 hours at approximately 20 °C. The non-dissolved test material was removed by filtration through a fine (0.22 μm) filter to give the 100.0 % saturated solution.
- The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae.
- Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd.
- Method of cultivation: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines. The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

The hardness of the test medium is 250 mg/L as CaCO3

Test temperature:

22.4 – 22.9 °C

pH:

7.15 – 8.31

Nominal and measured concentrations:

- Nominal: 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solution.
- Measured geometric mean concentrations: 2.08, 3.28, 5.75, 12.39 and 62.78 mg/L in presence of algae and 4.67, 9.11, 16.84, 41.22 and 85.17 mg/L in absence of algae
- Initial measured concentrations: 3.38, 7.95, 15.4, 39.2 and 80.7 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks covered with air-permeable stoppers.
- Material, size, headspace, fill volume: 100 mL
- Agitation: Continuously shaken by a laboratory orbital shaker to keep algae in suspension.
- Initial cells density: approximately 10^4 algal cells per mL test medium.
- No. of vessels per concentration: 3
- No. of vessels per vehicle control: 6
- There was an additional replicate without algae at each test concentration level for further analytical measurements (24, 48 and 72 hours) in order to
distinguish degradation and adsorption of the test material to algae.

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.
Separate stock solutions were first prepared in deionised water. The growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations:
- Stock solution 1 (macro nutrients): NH4Cl 15.0 mg/L, MgCl2.6H2O 12.0 mg/L, CaCl2.2H2O 18.0 mg/L, MgSO4.7H2O 15.0 mg/L and KH2PO4 1.6 mg/L.
- Stock solution 2 (iron): FeCl3.6H2O 64.0 µg/L and Na2EDTA.2H2O 100.0 µg/L.
- Stock solution 3 (trace elements): H3BO3 185.0 µg/L, MnCl2.4H2O 415.0 µg/L, ZnCl2 3.0 µg/L, CoCl2.6H2O 1.5 µg/L, CuCl2.2H2O 0.01 µg/L and Na2MoO4.2H2O 7.0 µg/L.
- Stock solution 4 (bicarbonate): NaHCO3 50.0 mg/L.
- Intervals of water quality measurement: Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The pH was checked at the beginning and at the end of the test, in the control and each concentration.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: The algal culture flasks were continuously illuminated.
- Light intensity and quality: The light intensity at the position occupied by algal culture flasks during the test was about 7813 lux (equivalent to 106 μE/m^2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Counting chamber
- The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. Microscopic observation of the algal cells in each concentration and in the control was performed (at 24, 48 and 72 h) to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Separation factor of 2.0
- Range finding study test concentrations: 0.1, 1, 10 and 100 % saturated solutions
A concentration range-finding test was conducted to determine the approximate toxicity of the test material so that appropriate test concentrations could be selected for use in the definitive test. Algal cells were exposed to each concentration of the test material plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three replicates in the control group.
- Results used to determine the conditions for the definitive study: Yes. Because significant inhibition was observed during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.0) and one control were used in the main test.

CALCULATIONS
- Calculation of Average Specific Growth Rate:
Concentration-effect relationship was calculated by comparing growth rates in control, test cultures in the following way. The average specific growth rate (μ) for individual cultures are calculated from the following relationship:

µ = [ln(Nn) - ln(N0)] / tn – t0

Where
ln (Nn) = natural logarithm of measured number of cells/mL at time tn
ln (N0) = natural logarithm of measured number of cells/mL at time t0
t0 = time (hour) of the beginning of the test
tn = time (hour) of nth measurements after the beginning of the test

The percentage inhibition of growth rate = % Iµ:

% Iµ= [(µc - µt) / µc ] ·100 %

Where
% Iμ = percent inhibition in average specific growth rate
μc = mean growth rate of the control
μt = mean growth rate of test concentration t

- Calculation of Area Under the Growth Curve:

A = [(N1 – N0) / 2] · t1 + [(N1 + N2 – 2N0) / 2] · (t2 – 21) + [(Nn-1 + Nn – 2N0) / 2] · (tn – tn – 1)

Where
N0 = nominal number of cells/mL at time t0 (start of the test)
N1 = mean measured number of cells/mL at t1 (24 hours)
N2 = mean measured number of cells/mL at t2 (48 hours)
Nn = mean measured number of cells/mL at tn
t1 = time of first measurement after start of the test
t2 = time of second measurement after start of the test
tn = time of nth measurement after start of the test

The percentage inhibition of area = % IA

% IA = [(Ac – At) / Ac] · 100 %

Where % IA = percent inhibition in area under the growth curve
Ac = mean area of the control
At = mean area of test concentration t

- Calculation of Yield:
Yield is calculated as the biomass at the end of the test minus the starting biomass for each single vessel of controls and treatments. For each test concentration and control, mean yield values were calculated.

Percentage inhibition in yield = % Iy

% Iy = [(yc – yi) / yc] · 100

Where:
yc = mean value for yield in the control group
yi = mean value for yield for the test concentration
Area under the growth curve (biomass), average specific growth rate and yield were calculated for each test flask. Then the mean area under the growth curve, the growth rate and mean yield were determined as arithmetic mean value over all test flasks per treatment.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

48.16 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. limits 39.50 – 58.72

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

7.95 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

biomass and yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

15.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

biomass and yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

16.37 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % conf. limits 14.61 – 18.35

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

18.99 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % conf. limits 16.83 – 21.44

Details on results:

CONCENTRATIONS OF THE TEST MATERIAL
- The test material could only be measured at the highest concentration level of 100.0 % saturated solution (nominal) 72 hours after the treatment (at the end of the experiment) in presence of algae.
- In order to calculate the mean exposure concentrations, where the measured concentration was below the Quantification Limit, the concentration was taken as half of the Limit of Quantification (LOQ = 1.0 mg/L). Therefore the corresponding measured geometric mean test material concentrations were: 2.08; 3.28; 5.75; 12.39 and 62.78 mg/L in presence of algae, while the measured geometric mean test material concentrations were: 4.67; 9.11; 16.84; 41.22 and 85.17 mg/L in absence of algae.
- According to the relevant guideline (OECD 201) disappearance of the test material from solution by absorption to the increasing algal biomass does not mean that it is lost from the test system. When the result of the test was analysed, it was checked whether a decrease in concentration of the test material in the course of the test was accompanied by a decrease in growth inhibition. If not, it may be appropriate to base the analysis of the results on initial (nominal or measured) concentrations.
- Based on these results most probably the test material is indeed absorbed by the increasing algal biomass, and should not be considered as lost from the test system. The specific growth rate for all of the tested concentrations was not clearly reduced at the section days 2-3 and it is indicating that a decrease of concentration of the test material was not clearly accompanied by a decrease in growth inhibition.
- Therefore it is recommended to express the EC50 and NOEC/LOEC values in nominal concentrations and not in the mean measured concentrations from the algae containing samples, thus biological results are related to the initial measured test material concentrations (3.38; 7.95; 15.4; 39.2 and 80.7 mg/L).

CELL NUMBERS
- The cell number in each flask (three replicates per treated group) was determined at the 24th, 48th, 72nd hours.

MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS
- Chuff cells were observed at the highest concentration level of 100.0 % saturated solution 48 and 72 hours after the treatment.

AVERAGE SPECIFIC GROWTH RATES
- The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h average specific growth rate was statistically significantly different from the untreated control value in the initial measured concentration range of 15.4 – 80.7 mg/L, correspondingly the No Observed Effect Concentration (NOEC) was determined as 7.95 mg/L (measured, initial).
- The 72 h ErC50 value was determined [by Probit analysis (TOXSTAT software)] as 48.16 mg/L (95 % confidence limits: 39.50 – 58.72 mg/L (measured, initial).

AREAS UNDER THE GROWTH CURVES
- The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h areas were statistically significantly different from the untreated control value in the initial measured concentration range of 15.4 – 80.7 mg/L, correspondingly the No Observed Effect Concentration (NOEC) was determined as 7.95 mg/L (measured, initial).
- The 72 h EbC50 value was determined [by Probit analysis (TOXSTAT software)] as 18.99 mg/L (95 % confidence limits: 16.83 – 21.44 mg/L (measured, initial).

YIELD
- The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h yield was statistically significantly different from the untreated control value in the initial measured concentration range of 15.4 – 80.7 mg/L, correspondingly the No Observed Effect Concentration (NOEC) was determined as 7.95 mg/L (measured, initial).
- The 72 h EyC50 value was determined [by Probit analysis (TOXSTAT software)] as 16.37 mg/L (95 % confidence limits: 14.61 – 18.35 mg/L (measured, initial).

Results with reference substance (positive control):

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate
satisfactory test conditions.
The 72h ErC 50: 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
The 72h EbC 50: 0.63 mg/L, (95 % confidence limits: 0.58 – 0.69 mg/L)
The 72h EyC 50: 0.53 mg/L, (95 % confidence limits: 0.49 – 0.58 mg/L)
These values are within the range of laboratory ring test data.

Reported statistics and error estimates:

The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0- 1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period.
The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
The ErC50, EbC50 and EyC50 values of the test material and their confidence limits were calculated using Probit analysis by TOXSTAT software (based on the calculated geometric mean concentrations).
Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software.
For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test.

Validity

- The cell density in the control cultures increased by a factor of 67.50 within three days.

- The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 7.17 %.

- The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.72 %.

- All validity criteria were met, therefore the study can be considered as valid.

 

Table 1: Growth Rates (μ) and Percentage Inhibition of μ during the Test Period

Concentration		Growth rate (μ) and % inhibition of μ
Nominal [% sat. sol.]	Measured [mg/L]	0-24 h		0-48 h		0-72 h
		µ	%	µ	%	µ	%
Control	0.00	0.0573	0.0	0.0586	0.0	0.0585	0.0
6.25	3.38	0.0569	0.8	0.0590	-0.7	0.0586	-0.2
12.5	7.95	0.0538	6.2	0.0590	-0.7	0.0585	-0.1
25.0	15.4	0.0529	7.8	0.0384*	34.5	0.0373*	36.3
50.0	39.2	0.0498	13.2	0.0384*	40.6	0.0328*	44.0
100.0	80.7	0.0345*	39.8	0.0173*	70.5	0.0240*	58.9

*: Statistically significantly different compared to the control values (Bonferroni t-Test; α = 0.05)

 

Table 2: Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Concentration		Area under the Growth Curves (A) and Percentage Inhibition of A
Nominal [% sat. sol.]	Measured [mg/L]	0-24 h		0-48 h		0-72 h
		A	%	A	%	A	%
Control	0.00	36.0	0.0	260.0	0.0	1246.0	0.0
6.25	3.38	36.0	0.0	264.0	-1.5	1260.0	-1.1
12.5	7.95	32.0	11.1	256.0	1.5	1248.0	-0.2
25.0	15.4	32.0	11.1	128.0*	50.8	356.0*	71.4
50.0	39.2	28.0	22.2	108.0*	58.5	276.0*	77.8
100.0	80.7	16.0*	55.6	48.0*	81.5	120.0*	90.4

*: Statistically significantly different compared to the control values (Bonferroni t-Test; α = 0.05)

 

Table 3: Yield (Y) and Percentage Inhibition of Y during the Test Period

Concentration		Yield
Nominal [% sat. sol.]	Measured [mg/L]	0-72 h
		Y	%
Control	0.00	66.5	0.0
6.25	3.38	67.0	-0.8
12.5	7.95	66.7	-0.3
25.0	15.4	13.7*	79.4
50.0	39.2	9.7*	85.5
100.0	80.7	4.7*	93.0

*: Statistically significantly different compared to the control values (Bonferroni t-Test; α = 0.05)

 

Measured Concentrations in the Absence of Algal Cells with the 95 % Confidence Intervals

Nominal Conc. [% Saturated Solution]	Measured Concentrations at the Start (mg/L)	Measured Concentrations After Day 1 (mg/L)	Measured Concentrations after 2 Days (mg/L)	Measured Concentrations at the End (mg/L)
Control	Not determined	Not determined	Not determined	Not determined
6.25	3.38 ± 0.523	4.24 ± 0.319	5.20 ± 0.164	6.37 ± 0.206
12.5	7.59 ± 0.178	8.33 ± 0.442	9.73 ± 1.864	10.7 ± 1.10
25.0	15.4 ± 0.66	15.5 ± 0.36	18.0 ± 0.51	18.7 ± 1.61
50.0	39.2 ± 1.64	38.4 ± 2.65	43.9 ± 2.82	43.7 ± 2.19
100.0	80.7 ± 3.68	80.9 ± 4.36	87.8 ± 3.81	91.8 ± 8.02

 

Measured Concentrations in Presence of Algal Cells with 95 % Confidence Intervals

Nominal Conc. [% Saturated Solution]	Measured Concentrations After Day 1 (mg/L)	Measured Concentrations after 2 Days (mg/L)	Measured Concentrations at the End (mg/L)
Control	Not determined	Not determined	Not determined
6.25	3.93 ± 0.713	2.81 ± 1.974	< LOQ
12.5	7.25 ± 0.722	3.89 ± 1.171	< LOQ
25.0	14.2 ± 0.72	9.99 ± 7.357	< LOQ
50.0	36.2 ± 2.48	33.2 ± 4.76	< LOQ
100.0	77.1 ± 3.54	73.2 ± 7.66	34.1 ± 9.93

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the initial measured concentration range of 15.4 – 80.7 mg/L. The overall NOEC was determined as 7.95 mg/L (measured, initial); the overall LOEC was determined as 15.4 mg/L (measured, initial). The 72-h EC50 values were 48.16, 16.37 and 18.99 mg/L (measured, initial) for growth rate, yield and biomass, respectively.

Executive summary:

The potential of the test material to cause aquatic toxicity to algae was examined in accordance with the standardised guidelines OECD 201, EU Method C.3., EPA OCSPP 850.5400 and JMAFF guidelines, under GLP conditions.

The effect of the test material was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.

A significant toxic response was observed during the preliminary range-finding test, therefore five test concentrations in a geometric series with a separation factor of 2.0 and one untreated control were tested in the main experiment.

The nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solution.

Test concentrations were analytically determined at the start of the test and at 24 hour intervals thereafter in order to better define loss of the test substance during the exposure period.

The test design included three replicates at each test concentration and six replicates for the untreated control. There was an additional replicate without algae at each test concentration level for further analytical measurements (24, 48 and 72 hours) in order to distinguish degradation and adsorption of the test material to algae.

According to the relevant guideline (OECD 201) disappearance of the test substance from solution by absorption to the increasing algal biomass does not mean that it is lost from the test system. When the result of the test was analysed, it was checked whether a decrease in concentration of the test substance in the course of the test was accompanied by a decrease in growth inhibition.

The corresponding measured geometric mean test material concentrations were: 2.08; 3.28; 5.75; 12.39 and 62.78 mg/L in presence of algae. In addition, results of the analytical measurements of samples in absence of algae showed measured geometric mean test material concentrations of 4.67; 9.11; 16.84; 41.22 and 85.17 mg/L for the nominal concentrations of 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solution, respectively.

Based on these results most probably the test material is indeed absorbed by the increasing algal biomass, and should not be considered as lost from the test system.

However based on the section-by-section average specific growth rates, it is indicating that a decrease of concentration of the test material was not clearly accompanied by a decrease in growth inhibition.

Therefore it is recommended to express the EC50 and NOEC/LOEC values in the initial test material concentrations (nominal or measured) and not in the mean measured concentrations from the algae containing samples, thus biological results are related to the initial measured test material concentrations (3.38; 7.95; 15.4; 39.2 and 80.7 mg/L).

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software. The ErC50, EbC50 and EyC50 values of the test material and their confidence limits were calculated using Probit analysis by TOXSTAT software.

Under the conditions of this study, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the initial measured concentration range of 15.4 – 80.7 mg/L. The overall NOEC was determined as 7.95 mg/L (measured, initial); the overall LOEC was determined as 15.4 mg/L (measured, initial). The 72-h EC50 values were 48.16, 16.37 and 18.99 mg/L (measured, initial) for growth rate, yield and biomass, respectively.

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2008/08/04-2008/09/05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with GLP and agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Signature: 15/10/2007; Date of Inspection: 21/08/2007

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Range finding test: 0.010, 0.10, 1.0, 10 and 100% v/v saturated solution
Definitive test: 6.25, 12.5, 25, 50 and 100% v/v sturated solution

- Sampling method:
Samples were taken for analysis following removal of any undissolved test material following filtration through a 0.2 μm Gelman Acrocap filter with the first 1 or 2 L being discarded.
24 h control: < LOQ
24 h filtered and 1 L discarded: 28.2 mg/L found.
24 h filtered and 2 L discarded: 27.7 mg/L found.

- Sample storage conditions before analysis:
Not applicable, Samples were analysed immediately after being taken.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Media preparation trial
Preliminary solubility work conducted indicated that the test material was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. Furthermore the test material was observed to form a precipitate upon addition to water.
Based on this information the test material was categorised as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test material under test conditions.

Saturated solution preparation
An amount of test material (1100 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C for 24 hours. After the stirring period samples were taken for chemical analysis after the following pre-treatments:
• Filtration through a 0.2 µm Sartorius Sartopore filter (approximately 1 litre discarded in order to pre-condition the filter)
• Filtration through a 0.2 µm Sartorius Sartopore filter (approximately 2 litres discarded in order to pre-condition the filter)

- Eluate:
Culture medium
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l
The culture medium was prepared using reverse osmosis purified deionised water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

- Differential loading:
Not applicable.

- Controls:
Range finding test:
Blank control: 2 replicates

Definitive test:
Blank control: 6 replicates

Positive control:
Blank control: 6 replicates
Positive control concentrations: 0.0625, 0.125, 0.25, 0.50, 1.0 mg/l (3 replicates each concentration)

- Chemical name of vehicle:
Not applicable.

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
Not applicable.

- Evidence of undissolved material (e.g. precipitate, surface film, etc):
At the start of the test all control, 6.25 and 12.5% v/v saturated solution test cultures were observed to be clear colourless solutions whilst the 25, 50 and 100% v/v saturated solution test cultures were observed to be clear colourless solutions with a slight foam at the surface. After the 72-Hour test period all control, 6.25, 12.5 and 25% v/v saturated solution test cultures were observed to be pale green dispersions. The 50% v/v saturated solution test cultures were observed to be very pale green dispersions whilst the 100% v/v saturated solution test cultures were observed to be slightly hazy dispersions.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name:
Green algae.

- Strain:
CCAP 276/20

- Source (laboratory, culture collection):
Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.

- Age of inoculum (at test initiation):
Not applicable.

- Method of cultivation:
Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (same as test culture medium). The master cultures were maintained in the laboratory under constant aeration and constant illumination at 24 ± 1 deg C.

ACCLIMATION
- Acclimation period:
Not applicable.

- Culturing media and conditions (same as test or not):
Same as test.

- Any deformed or abnormal cells observed:
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Not stated.

Hardness:

Not stated.

Test temperature:

24 ± 1 deg C

pH:

7.3 - 7.4 at 0 hours
7.8 - 8.3 at 72 hours

Dissolved oxygen:

Not stated.

Salinity:

Not applicable.

Nominal and measured concentrations:

Testing was conducted upon serial dilutions of a saturated solutions, therefore nominal concentrations are expressed as a % of the saturated solution.
the 100% saturated solution was found to be 32mg/l by HPLC, however due to serial dilutions forming precipitate concentrations lower than 100% were below the limit of quantification of the analytical equipment.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type:
closed

- Material, size, headspace, fill volume:
250ml glass conical flasks each containing 100ml of test preparation.

- Aeration:
Not applicable.

- Type of flow-through:
Not applicable.

- Renewal rate of test solution:
Not applicable

- Initial cells density:
4x 10^3 cells/ml.

- Control end cells density:
Mean cell density: 2.29 x 10^5 cells per ml.

- No. of organisms per vessel:
Not stated.

- No. of vessels per concentration (replicates):
Range finding test: 2 replicates to each concentration
Definitive test: 3 replicates to each concentration

- No. of vessels per control (replicates):
Range finding test: 2 replicates
Definitive test: 6 replicates

- No. of vessels per vehicle control (replicates):
Not applicable, no vehicle used.

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used:
NaNO3 25.5 mg/L
MgCl2.6H2O 12.165 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H20 14.7 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.1855 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.159 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
Na2SeO3.5H2O 0.000010 mg/L
The culture medium was prepared using reverse osmosis purified water and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCl.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Test medium was the same as the culture medium.

- Total organic carbon:
Not stated.

- Particulate matter:
Not stated.

- Metals:
Not stated.

- Pesticides:
Not stated.

- Chlorine:
Not stated.

- Alkalinity:
Not stated.

- Ca/mg ratio:
Not stated.

- Conductivity:
Not stated.

- Culture medium different from test medium:
The culture medium was the same as the test medium

- Intervals of water quality measurement:
Not applicable.

OTHER TEST CONDITIONS
- Sterile test conditions:
Yes

- Adjustment of pH:
The culture medium was prepared using reverse osmosis purified deionised water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
*Elga Optima 15+ or Elga Purelab Option R-15 BP

- Photoperiod:
Constant illumination.

- Light intensity and quality:
Approximately 7000 lux provided by warm lighting (380-730nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
Samples were taken at 0 and 25, hours and the cell densities determined using a Coulter® Multisizer Particle Counter. The presence of particulate matter in the test cultures at 47 hours interfered with the Coulter counts. It was therefore considered appropriate to determine algal cell densities at 47 and 72 hours using a haemocytometer and light microscope in order to give an accurate count of the number of algal cells present.


TEST CONCENTRATIONS
- Spacing factor for test concentrations:
Range finding test: 10
Definitive test: 2

- Justification for using less concentrations than requested by guideline:
Not applicable.

- Range finding study
- Test concentrations:
0.010, 0.10, 1.0, 10, 100 % saturated solution.

- Results used to determine the conditions for the definitive study:
Yes.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

50 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control:
yes

- Observation of abnormalities:
- Unusual cell shape:
No abnormalities recorded.

- Colour differences:
At the start of the test all control, 6.25 and 12.5% v/v saturated solution test cultures were observed to be clear colourless solutions whilst the 25, 50 and 100% v/v saturated solution test cultures were observed to be clear colourless solutions with a slight foam at the surface. After the 72-Hour test period all control, 6.25, 12.5 and 25% v/v saturated solution test cultures were observed to be pale green dispersions. The 50% v/v saturated solution test cultures were observed to be very pale green dispersions whilst the 100% v/v saturated solution test cultures were observed to be slightly hazy dispersions.

- Flocculation:
Not stated.

- Adherence to test vessels:
Not stated.

- Aggregation of algal cells:
Not stated.

- Other:

- Any stimulation of growth found in any treatment:
None recorded.

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
Preliminary solubility work conducted indicated that the test material was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. Furthermore the test material was observed to form a precipitate upon addition to water.
Particulate matter was observed in the test concentrations at 72 hours.

- Effect concentrations exceeding solubility of substance in test medium:
Not applicable.

Range-Finding Test
- The results showed no effect on growth at the test concentrations of 0.01, 0.1, 1.0 and 10 % v/v saturated solution. However, growth was observed to be reduced at 100 % v/v saturated solution.

Results with reference substance (positive control):

- Results with reference substance valid?
Yes

- EC50:
ErC50 at 72 hours 0.57 mg/l, 95% confidence limits 0.48 - 0.66 mg/l
EyC50 at 72 hours 0.32 mg/l, 95% confidence limits 0.29 - 0.35 mg/l
EbC50 at 72 hours 0.31 mg/l, 95% confidence limits 0.28 - 0.35 mg/l

Exposure of Desmodesmus subspicatus to the reference material, potassium dichromate, gave an ErC50(0 - 72 h) of 0.57 mg/l; 95% confidence limits 0.48 – 0.66 mg/l, an EyC50 (0 - 72 h) of 0.32 mg/l; 95% confidence limits 0.29 - 0.35 mg/l, and an EbC50 (0 - 72 h) of 0.31 mg/l; 95% confidence limits 0.28 - 0.35 mg/l. The Lowest Observed Effect Concentrations based on inhibition of growth rate, yield and biomass integral were 0.50, 0.125 and 0.125 mg/l respectively and the No Observed Effect Concentrations were 0.25, 0.0625 and 0.0625 mg/l respectively.

- Other:
A positive control used potassium dichromate as the reference material. An amount of reference material (100 mg) was dissolved in culture medium and the volume adjusted to 1 litre to give a 100 mg/l stock solution from which a series of dilutions were made to give further stock solutions of 10, 2.0, 1.0, 0.50, 0.25 and 0.125 mg/l. An aliquot (250 ml) of each of the 0.125, 0.25, 0.50, 1.0 and 2.0 mg/l stock solutions was separately mixed with algal suspension (250 ml) to give the required test concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.
The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Six replicate flasks were prepared for the control and three replicate flasks prepared for each test concentration.
The flasks were incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Reported statistics and error estimates:

See remarks section.

 Evaluation of data

 Comparison of growth rates

The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the equation:

µ = (ln N_n - ln N₁)/(t_n - t₁)

where:

µ         =         average specific growth rate from time t₁to t_n

N₁       =         cell concentration at t₁

N_n       =         cell concentration at t_n

t₁         =         time of first measurement

t_n         =         time of n^th measurement

The average specific growth rate over the test duration was calculated for each replicate control and test material vessel using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.

In addition the section by section specific growth rate (days 0-1, 1-2 and 2-3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.

Percentage inhibition of growth rate for each replicate test material vessel was calculated using the following equation:

Ir = (µ_c - µ_t)/µ_cx100

where:

I_r         =         percentage inhibition of average specific growth rate

µ_c        =         mean average specific growth rate for the control cultures

µ_t        =         average specific growth rate for the test culture

Comparison of Yield

Yield is calculated as the increase in biomass over the exposure period using the following equation:

Y = N_n - N₀

where:

Y        =         yield

N₀       =         cell concentration at the start of the test

N_n       =         cell concentration at the end of the test

For each test concentration and control the mean value for yield along with the standard deviation was calculated. Percentage inhibition of yield was calculated using the following equation:

I_y = (Y_c - Y_t)/Y_cx100

where:

I_y         =         percentage inhibition of yield

Y_c       =         mean value for yield in the control group

Y_t        =         mean value for yield for the treatment group

Comparison of biomass integral

The biomass integral (area under the growth curve) was calculated using the following equation:

A = ((N₁ - N₀)/2) x t₁ + ((N₁ + N₂ - 2N₀)/2) x (t₂ - t₁) + ((N_n-1 + N_n - 2N₀)/2) x (t_n - t_n-1)

where:

A        =         area

N₀       =         nominal cell concentration at start of test

N₁       =         measured cell concentration at t₁

N_n       =         measured cell concentration at t_n

t₁         =         time of first measurement after beginning of test

t_n         =         time of n^thmeasurement after beginning of test

Percentage inhibition of the biomass integral for each replicate test material vessel was calculated using the following equation:

I_A = ((A_c - A_t)/A_c) x 100

where:

I_A        =         percentage inhibition of the biomass integral

A_c       =         mean biomass integral for the control cultures

A_t        =         biomass integral for the test culture

Determination of EC_xvalues

For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package (IDBS). EC_xvalues were then determined from the equation for the fitted line.

Where appropriate 95% confidence limits for the EC₅₀values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949). 

Statistical analysis

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) was carried out on the growth rate, yield and biomass integral data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001).

Validity criteria fulfilled:

yes

Remarks:

The study fullfills all the validity endpoints as specified by the OECD guideline.

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave an ErC50 (0 - 72 h) of 100% v/v saturated solution, an EyC50 (0 - 72 h) of 51% v/v saturated solution; 95% confidence limits 44 – 61% v/v saturated solution, and an EbC50 (0 - 72 h) of 64% v/v saturated solution; 95% confidence limits 57 – 71% v/v saturated solution. The Lowest Observed Effect Concentration based on growth rate, yield and biomass integral was 50% v/v saturated solution, and the No Observed Effect Concentration was 25% v/v saturated solution.
It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

Executive summary:

Introduction.

A study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods. 

Preliminary solubility work conducted indicated that the test material was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing. A pre-study media preparation trial indicated that a dissolved test material concentration of approximately 28 mg/l was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to solutions of the test material at nominal concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test material solutions were prepared by stirring an excess (100 mg/l) of test material in culture medium using a propeller stirrer at approximately 1500 rpm at a temperature of approximately 21°C for 24 hours. After the stirring period any undissolved test material was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test material. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using either a Coulter^®Multisizer Particle Counter or a haemocytometer and light microscope.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Desmodesmus subspicatus was exposed to an aqueous solution of the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

Results.  

In terms of growth rate, exposure of Desmodesmus subspicatus to the test material gave an E_rC₅₀(0 - 72 h) value of 100% v/v saturated solution. The Lowest Observed Effect Concentration based on inhibition of growth rate was 50% v/v saturated solution and the No Observed Effect Concentration was 25% v/v saturated solution.

In terms of yield, exposure of Desmodesmus subspicatus to the test material gave an E_yC₅₀(0 - 72 h) value of 51% v/v saturated solution; 95% confidence limits 44 – 61% v/v saturated solution. The Lowest Observed Effect Concentration based on yield was 50% v/v saturated solution and the No Observed Effect Concentration was 25% v/v saturated solution.

In terms of biomass integral (area under growth curve), exposure of Desmodesmus subspicatus to the test material gave an E_bC₅₀(0 - 72 h) value of 64% v/v saturated solution; 95% confidence limits 57 – 71% v/v saturated solution. The Lowest Observed Effect Concentration based on inhibition of biomass integral was 50% v/v saturated solution and the No Observed Effect Concentration was 25% v/v saturated solution.

Chemical analysis of the saturated solution at 0 hours showed a measured test concentration of 32 mg/l was obtained. However, subsequent dilutions performed from this saturated solution showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained. During the range-finding test it was noted that the test material formed a precipitate in culture medium. Based on the structure of the test material it was considered that it was forming insoluble complexes with the magnesium and calcium ions present in the test media. During the 24-Hour saturated solution preparation period the test material had reacted with all of the calcium and magnesium ions present in the culture medium. Whilst filtration of the saturated solution removed any undissolved test material and/or precipitate present, serial dilutions performed in fresh culture medium to provide the remaining test concentrations caused further precipitation of the test material to occur. Given that the method of chemical analysis employed (high performance liquid chromatography (HPLC)) only detects the soluble fractions present in aqueous test samples this would account for measured concentrations of less than the LOQ being obtained. 

Analysis of the test preparations at 72 hours showed measured test concentrations of less than the LOQ were obtained for all test concentrations employed. This decline was inline with the preliminary stability analyses conducted which indicated that the test material was unstable in culture medium over the 72-Hour test period. 

Given that the concentrations of the test material could not be determined in the test media (with the exception of the saturated solution) the results are based on concentrations as % v/v dilution of the saturated solution only. 

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

See the read-across report attached in Section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.51 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CL = 1.5-2.2 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: CL = 1.6-2.0 mg/L

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.92 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

See the read-across report attached in Section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

48.16 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. limits 39.50 – 58.72

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

7.95 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

biomass and yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

15.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

biomass and yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

16.37 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % conf. limits 14.61 – 18.35

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

18.99 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % conf. limits 16.83 – 21.44

Description of key information

Key Study on Read Across Substance HS-21P-T: Vryenhoef & Mullee (2009)
Based on the geometric mean measured test concentrations the ErC50 (0 - 72 h) value was 4.3 mg/L, the EyC50 (0 - 72 h) value was 1.8 mg/L; 95% confidence limits 1.5 – 2.2 mg/L and the EbC50 (0 - 72 h) value was 1.8 mg/L; 95% confidence limits 1.6 – 2.0 mg/L. The Lowest Observed Effect Concentration based on growth rate, yield and biomass integral was 0.92 mg/L, and the No Observed Effect Concentration was 0.51 mg/L.

Supporting Study on Read Across Substance GS-11P-T: Sipos (2018)
Under the conditions of this study, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the initial measured concentration range of 15.4 – 80.7 mg/L. The overall NOEC was determined as 7.95 mg/L (measured, initial); the overall LOEC was determined as 15.4 mg/L (measured, initial). The 72-h EC50 values were 48.16, 16.37 and 18.99 mg/L (measured, initial) for growth rate, yield and biomass, respectively.

Supporting Study on Target Substance: Vryenhoef & Mullee (2008) 
72 h ErC50 100 % v/v saturated solution (equivalent to 32 mg/L) LOEC 50 % v/v saturated solution and NOEC 25 % v/v saturated solution, OECD 201, EU Method C.3.

Key value for chemical safety assessment

EC50 for freshwater algae:

4.3 mg/L

EC10 or NOEC for freshwater algae:

0.51 mg/L

Additional information

Supporting Study on Target Substance: Vryenhoef & Mullee (2008) 

In the key study Vryenhoef & Mullee 2008, the effect of the test material on the growth of the green alga Desmodesmus subspicatus was studied. The method followed that described in the OECD 201 and EU Method C.3. The study was performed under GLP conditions. The study was therefore assigned a reliability score of 1 in accordance with the principles for assessing data quality as described in Klimisch et al. (1997).

Preliminary solubility work conducted indicated that the test material was practically insoluble in water. A pre-study media preparation trial indicated that a dissolved test material concentration of approximately 28 mg/L was obtained from a saturated solution method of preparation, indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to solutions of the test material at nominal concentrations of 6.25, 12.5, 25, 50 and 100 % v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test material solutions were prepared by stirring an excess (100 mg/L) of test material in culture medium using a propeller stirrer at approximately 1500 rpm at a temperature of approximately 21 °C for 24 hours. After the stirring period any undissolved test material was removed by filtration to produce a 100 % v/v saturated solution of the test material. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using either a Coulter^® Multisizer Particle Counter or a haemocytometer and light microscope. The test material gave an E_rC₅₀(0 - 72 h) value of 100 % v/v saturated solution. The Lowest Observed Effect Concentration based on inhibition of growth rate was 50 % v/v saturated solution and the No Observed Effect Concentration was 25 % v/v saturated solution.

The test material gave an E_yC₅₀(0 - 72 h) value of 51 % v/v saturated solution; 95 % confidence limits 44 – 61 % v/v saturated solution. The Lowest Observed Effect Concentration based on yield was 50 % v/v saturated solution and the No Observed Effect Concentration was 25 % v/v saturated solution.

The biomass integral (area under growth curve) from exposure of the algae to the test material gave an E_bC₅₀(0 - 72 h) value of 64 % v/v saturated solution; 95 % confidence limits 57 – 71 % v/v saturated solution. The Lowest Observed Effect Concentration based on inhibition of biomass integral was 50 % v/v saturated solution and the No Observed Effect Concentration was 25 % v/v saturated solution.

Chemical analysis of the saturated solution at 0 hours showed a measured test concentration of 32 mg/L was obtained. However, subsequent dilutions performed from this saturated solution showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained. Given that the concentrations of the test material could not be determined in the test media (with the exception of the saturated solution) the results are based on concentrations as % v/v dilution of the saturated solution only.

 

Key Study on Read Across Substance HS-21P-T: Vryenhoef & Mullee (2009)

In the key study on target substance HS-21P-T, Vryenhoef & Mullee (2009), the study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC). The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

 Whilst the test material was observed to be readily soluble in water, upon addition to culture medium the test material was observed to form a precipitate. A pre-study media preparation trial indicated that a dissolved test material concentration of approximately 51 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to solutions of the test material at 0-Hour measured concentrations of 0.54, 1.3, 4.0, 14 and 42 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test material solutions were prepared by stirring an excess (100 mg/L) of test material in culture medium using a propeller stirrer at approximately 1500 rpm at a temperature of approximately 21°C for 24 hours. After the stirring period any undissolved test material was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a saturated solution of the test material with a 0-Hour measured concentration of 42 mg/L. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using either a Coulter^® Multisizer Particle Counter or a haemocytometer and light microscope.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Desmodesmus subspicatus was exposed to an aqueous solution of the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^® Multisizer Particle Counter.

In terms of growth rate, exposure of Desmodesmus subspicatus to the test material gave an E_rC₅₀(0 - 72 h) value of 34 mg/L*. The Lowest Observed Effect Concentration based on inhibition of growth rate was 4.0 mg/L and the No Observed Effect Concentration was 1.3 mg/L.

In terms of yield, exposure of Desmodesmus subspicatus to the test material gave an E_yC₅₀ (0 - 72 h) value of 14 mg/L; 95% confidence limits 11 - 18 mg/L. The Lowest Observed Effect Concentration based on yield was 4.0 mg/L and the No Observed Effect Concentration was 1.3 mg/L.

In terms of biomass integral (area under growth curve), exposure of Desmodesmus subspicatus to the test material gave an E_bC₅₀(0 - 72 h) value of 13 mg/L; 95% confidence limits 11 - 16 mg/L. The Lowest Observed Effect Concentration based on inhibition of biomass integral was 4.0 mg/L and the No Observed Effect Concentration was 1.3 mg/L.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.54 to 42 mg/L. A decline in measured test concentrations in the range of less than the limit of quantitation (LOQ) of the analytical method employed which was determined to be 0.41 mg/L to 0.66 mg/L was observed at 72 hours. This decline was inline with the preliminary stability analyses conducted which indicated that the test material was unstable in culture medium over the test duration.

Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. The E_rC₅₀(0 - 72 h) based on the geometric mean measured test concentrations was 4.3 mg/L*, the E_yC₅₀(0 - 72 h) was 1.8 mg/L; 95% confidence limits 1.5 – 2.2 mg/L, and the    E_bC₅₀(0 - 72 h) was 1.8 mg/L; 95% confidence limits 1.6 – 2.0 mg/L. The Lowest Observed Effect Concentration based on inhibition of growth rate, yield and biomass integral was 0.92 mg/L and the No Observed Effect Concentration was 0.51 mg/L.

Exposure of Desmodesmus subspicatus to the reference material, potassium dichromate, gave an E_rC₅₀(0 - 72 h) of 0.57 mg/L; 95% confidence limits 0.48 – 0.66 mg/L, an E_yC₅₀(0 - 72 h) of 0.32 mg/L; 95% confidence limits 0.29 ‑ 0.35 mg/L, and an E_bC₅₀(0 - 72 h) of 0.31 mg/L; 95% confidence limits 0.28 - 0.35 mg/L. The Lowest Observed Effect Concentrations based on inhibition of growth rate, yield and biomass integral were 0.50, 0.125 and 0.125 mg/L respectively and the No Observed Effect Concentrations were 0.25, 0.0625 and 0.0625 mg/L respectively. 

*It was not possible to calculate 95% confidence limits for the E_rC₅₀value as the data generated did not fit the models available for the calculation of confidence limits.

 

Supporting Study on Read Across Substance GS-11P-T: Sipos (2018)

In the supporting study on target substance GS-11P-T Sipos (2018), the potential of the test material to cause aquatic toxicity to algae was examined in accordance with the standardised guidelines OECD 201, EU Method C.3., EPA OCSPP 850.5400 and JMAFF guidelines, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The effect of the test material was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.

A significant toxic response was observed during the preliminary range-finding test, therefore five test concentrations in a geometric series with a separation factor of 2.0 and one untreated control were tested in the main experiment.

The nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solution.

Test concentrations were analytically determined at the start of the test and at 24 hour intervals thereafter in order to better define loss of the test substance during the exposure period.

The test design included three replicates at each test concentration and six replicates for the untreated control. There was an additional replicate without algae at each test concentration level for further analytical measurements (24, 48 and 72 hours) in order to distinguish degradation and adsorption of the test material to algae.

According to the relevant guideline (OECD 201) disappearance of the test substance from solution by absorption to the increasing algal biomass does not mean that it is lost from the test system. When the result of the test was analysed, it was checked whether a decrease in concentration of the test substance in the course of the test was accompanied by a decrease in growth inhibition.

The corresponding measured geometric mean test material concentrations were: 2.08; 3.28; 5.75; 12.39 and 62.78 mg/L in presence of algae. In addition, results of the analytical measurements of samples in absence of algae showed measured geometric mean test material concentrations of 4.67; 9.11; 16.84; 41.22 and 85.17 mg/L for the nominal concentrations of 6.25, 12.5, 25.0, 50.0 and 100.0 % saturated solution, respectively.

Based on these results most probably the test material is indeed absorbed by the increasing algal biomass, and should not be considered as lost from the test system.

However based on the section-by-section average specific growth rates, it is indicating that a decrease of concentration of the test material was not clearly accompanied by a decrease in growth inhibition.

Therefore it is recommended to express the EC50 and NOEC/LOEC values in the initial test material concentrations (nominal or measured) and not in the mean measured concentrations from the algae containing samples, thus biological results are related to the initial measured test material concentrations (3.38; 7.95; 15.4; 39.2 and 80.7 mg/L).

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software. The ErC50, EbC50 and EyC50 values of the test material and their confidence limits were calculated using Probit analysis by TOXSTAT software.

Under the conditions of this study, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the initial measured concentration range of 15.4 – 80.7 mg/L. The overall NOEC was determined as 7.95 mg/L (measured, initial); the overall LOEC was determined as 15.4 mg/L (measured, initial). The 72-h EC50 values were 48.16, 16.37 and 18.99 mg/L (measured, initial) for growth rate, yield and biomass, respectively.