C.I. Reactive Yellow 2

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 October 2016 to 27 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: 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, Ministry of Health, Labour and Welfare and Ministry of the Environment.

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 (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:

- Concentrations: Analytical measurement was performed at the applied test concentration levels (all concentrations) and from the control at the beginning and at the end of the experiment.
- Sampling method: The samples were analyzed by HPLC-UV method.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution with a nominal concentration of 100 mg/L was prepared with direct addition of the test item, mixed into algal growth medium (OECD Medium). The test solutions were prepared by appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae. See Table 1 for the preparation of test solutions from stock solution.
- Untreated control: Algal growth medium was inoculated with algal cells (without test item) and was examined in parallel to the test item concentrations.
- Reference 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.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable, the test material was added to OECD medium.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- 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 in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd.
- Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
- Method of cultivation/Breeding conditions: 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

Test temperature:

The temperature was in the range of 22.8 – 23.2 °C measured in the flask and between 22.7 and 23.7 °C measured within the climate chamber.

pH:

The range of the pH was 7.41 – 8.50 during the experiment.

Nominal and measured concentrations:

- The nominal concentrations in the definitive test were the following: 6.25, 12.5, 25, 50, and 100 mg/L.
- The corresponding measured geometric mean test item concentrations were: 6.32; 12.85; 25.55; 50.95 and 103.5 mg/L.
As the measured concentrations deviated not more than 20 per cent from the nominal in all cases, the biological results are based on the nominal test item concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type: Closed. The flasks were covered with air-permeable stoppers.
- Material, size, headspace, fill volume: Volumes of 100 mL algal suspension per replicate
- Aeration: Not specified. Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension.
- No. of organisms per vessel: 10^4 algal cells per mL test medium
- No. of vessels per concentration: Three replicates per test concentration
- No. of vessels per control: Six replicates in the control group

GROWTH MEDIUM
- Standard medium used: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests. See Table 2 for composition of OECD medium.

OTHER TEST CONDITIONS
- Adjustment of pH: No. The pH was checked at the beginning and at the end of the test, in the control and each concentration. The pH of the control medium was not increased by more than 1.5 units during the test.
- 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 7905 lux (equivalent to ~107 μE/m2/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:
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 h, 48 h and 72 h) to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Range finding study: Yes. Algal cells were exposed to each concentration of the test item plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three replicates in the control group.
- Test concentrations: The concentration levels used and results (72 h) of the preliminary range-finding test were 0.1, 1.0, 10.0 and 100.0 mg/L
- Results used to determine the conditions for the definitive study: Yes. The concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations could be selected for use in the definitive test.
Thin cells could be observed at the highest concentrations of 50 and 100 mg/L (nominal).
Because slight inhibition was observed at the highest concentration level during the preliminary range-finding test, five test concentrations in a geometric series (factor 2.0) and one control were used in the main experiment.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS
Thin cells were observed at the two highest examined concentrations of 50 and 100 mg/L (nominal) during the experiment.

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 concentration range of 25 - 100 mg/L (nominal), but the observed effects (inhibition: μ = 2.2, 3.0 and 8.3 % at 25, 50 and 100 mg/L, respectively) are not biologically significant, but are due to the biological variability of the test system. However, due to that thin cells were observed at the two highest nominal concentrations of 50 and 100 mg/L during the experiment and it is not negligible and should also be considered, therefore the No Observed Effect Concentration (NOEC) determined as 25 mg/L (nominal).
The 72 h ErC50 was determined to be higher than 100 mg/L (nominal), the highest concentration tested.

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 concentration range of 25 - 100 mg/L (nominal), but the observed effect (inhibition: A = 10.1 %) at 25 mg/L is not biologically significant, but is due to the biological variability of the test system. Therefore the No Observed Effect Concentration (NOEC) determined as 25 mg/L (nominal). The 72 h EbC50 value was determined to be higher than 100 mg/L (nominal), the highest concentration tested.

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 tested concentration range of 25 - 100 mg/L (nominal), but the observed effect (inhibition: Y = 9.0 %) at 25 mg/L is not biologically significant, but is due to the biological variability of the test system. Therefore the No Observed Effect Concentration (NOEC) determined as 25 mg/L (nominal). The 72 h EyC50 was determined to be higher than 100 mg/L (nominal), the highest concentration tested.
Even if statistically significant effect was calculated at the tested concentration levels of 25, 50 and 100 mg/L (nominal) in case of growth rates, however the observed effects (μ = 2.2, 3.0 and 8.3 %, respectively) are not biologically significant, but are due to the biological variability of the test system. However thin cells were observed at the two highest nominal concentrations of 50 and 100 mg/L during the experiment and it is not negligible and should also be considered.
Although statistically significant effect was calculated at the tested nominal concentration level of 25 mg/L in case of biomass and yield, however the observed effects (A = 10.1 %, Y = 9.0 %) are not biologically significant, but are due to the biological variability of the test system.
Taking into account description above the overall NOEC was determined as 25 mg/L (nominal) and the overall LOEC could be determined as 50 mg/L (nominal).

Results with reference substance (positive control):

Reference Control: Potassium dichromate
The 72h ErC 50: 0.89 mg/L, (95 % confidence limits: 0.82 – 0.97 mg/L)
The 72h EbC 50: 0.53 mg/L, (95 % confidence limits: 0.49 – 0.58 mg/L)
The 72h EyC 50: 0.62 mg/L, (95 % confidence limits: 0.57 – 0.67 mg/L)

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.
The ErC50, EbC50 and EyC50 values of the test item could not be calculated.
Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of varianc (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.

Table 3: Growth Rates (μ) and Percentage Inhibition of μ during the Test Period

Nominal Concentration [mg/L]	Growth rate (μ) and % inhibition of μ
	0–24 h		0–48 h		0–72 h
	μ	%	μ	%	μ	%
Control	0.0593	0.0	0.0613	0.0	0.0596	0.0
6.25	0.0569	4.1	0.0606	1.1	0.0593	0.4
12.5	0.0538	9.3	0.0594	3.1	0.0588	1.3
25	0.0538	9.3	0.0590	3.7	0.0583*	2.2
50	0.0609	-2.6	0.0586	4.4	0.0578*+	3.0
100	0.0538	9.3	0.0573*	6.5	0.0546*	8.3

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

+ : at these values the rounding of the EXCEL and TOXSTAT software was different. The table contains the values calculated with EXCEL.

Validity

The cell density in the control cultures increased by the factor of 72.83 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 9.02 %.

The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.80 %.

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

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, EC50 for growth rate (ErC50) was > 100 mg/L (nominal). The NOEC for growth rate was 25 mg/L (nominal) and the LOEC was 50 mg/L (nominal).

Executive summary:

The acute toxicity of the test material to the freshwater alga was investigated in a study performed under GLP conditions in accordance to the standardised guideline OECD 201, EU Method C.3 and the Japanese guidelines YAKUSHOKHATSU No. 1121002, SEIKYOKU No.2 and KANPOKIHATSU No. 021121002 and JMAFF 2-7-7.

Because slight inhibition was observed during the preliminary concentration range-finding test, therefore five test concentrations in a geometric series (factor 2.0) and one control were tested in the definitive test.

The nominal concentrations of test item used in the main experiment were: 6.25, 12.5, 25, 50, and 100 mg/L.

The test concentration was analytically determined at the start and at the end of the experiment. The corresponding measured geometric mean test item concentrations were 6.32; 12.85; 25.55; 50.95 and 103.5 mg/L.

As the analytically measured concentration deviated not more than 20 per cent from the nominal, the biological results are based on the nominal test item concentration.

The test design included three replicates at each test concentration and six replicates for the untreated controls.

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. Furthermore values were biologically significantly different compared to the control were also considered.

The ErC50, EbC50 and EyC50 values of the test item could not be calculated and determined directly from the raw data.

Even if a statistically significant effect was calculated at the tested concentration levels of 25, 50 and 100 mg/L (nominal) in case of growth rates, however the observed effects (μ = 2.2, 3.0 and 8.3 %, respectively) are not biologically significant, but are due to the biological variability of the test system. However thin cells were observed at the two highest nominal concentrations of 50 and 100 mg/L during the experiment and it is not negligible and should also be considered.

Although a statistically significant effect was calculated at the tested nominal concentration level of 25 mg/L in case of biomass and yield, however the observed effects (A = 10.1 %, Y = 9.0 %) are not biologically significant, but are due to the biological variability of the test system.

The overall NOEC was determined as 25 mg/L (nominal) and the overall LOEC could be determined as 50 mg/L (nominal).

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the examined concentration range of 50 - 100 mg/L (nominal).

Under the conditions of the study, the overall NOEC was determined as 25 mg/L (nominal) and the overall LOEC was determined as 50 mg/L (nominal).

Description of key information

Under the conditions of the study, EC50 for growth rate (ErC50) was > 100 mg/L (nominal). The NOEC for growth rate was 25 mg/L (nominal) and the LOEC was 50 mg/L (nominal).

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

Additional information

The acute toxicity of the test material to the freshwater alga was investigated in a study performed under GLP conditions in accordance to the standardised guideline OECD 201, EU Method C3 and the Japanese guidelines YAKUSHOKHATSU No. 1121002, SEIKYOKU No.2 and KANPOKIHATSU No. 021121002 and JMAFF 2-7-7. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The acute toxicity of the test material to the freshwater alga was investigated in a study performed under GLP conditions in accordance to the standardised guideline OECD 201, EU Method C.3 and the Japanese guidelines YAKUSHOKHATSU No. 1121002, SEIKYOKU No.2 and KANPOKIHATSU No. 021121002 and JMAFF 2-7-7.

Because slight inhibition was observed during the preliminary concentration range-finding test, therefore five test concentrations in a geometric series (factor 2.0) and one control were tested in the definitive test.

The nominal concentrations of test item used in the main experiment were: 6.25, 12.5, 25, 50, and 100 mg/L.

The test concentration was analytically determined at the start and at the end of the experiment. The corresponding measured geometric mean test item concentrations were 6.32; 12.85; 25.55; 50.95 and 103.5 mg/L.

As the analytically measured concentration deviated not more than 20 per cent from the nominal, the biological results are based on the nominal test item concentration.

The test design included three replicates at each test concentration and six replicates for the untreated controls.

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. Furthermore values were biologically significantly different compared to the control were also considered.

The ErC50, EbC50 and EyC50 values of the test item could not be calculated and determined directly from the raw data.

Even if a statistically significant effect was calculated at the tested concentration levels of 25, 50 and 100 mg/L (nominal) in case of growth rates, however the observed effects (μ = 2.2, 3.0 and 8.3 %, respectively) are not biologically significant, but are due to the biological variability of the test system. However thin cells were observed at the two highest nominal concentrations of 50 and 100 mg/L during the experiment and it is not negligible and should also be considered.

Although a statistically significant effect was calculated at the tested nominal concentration level of 25 mg/L in case of biomass and yield, however the observed effects (A = 10.1 %, Y = 9.0 %) are not biologically significant, but are due to the biological variability of the test system.

The overall NOEC was determined as 25 mg/L (nominal) and the overall LOEC could be determined as 50 mg/L (nominal).

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the examined concentration range of 50 - 100 mg/L (nominal).

Under the conditions of the study, the overall NOEC was determined as 25 mg/L (nominal) and the overall LOEC was determined as 50 mg/L (nominal).