Reaction mass of hydrogen [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1) and hydrogen bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1) and hydrogen bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

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:

26 September 2017 to 29 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2011

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)

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:

- The test material was measured in the test concentration (100 mg/L nominal loading rates WAF) at the start and end of the experiment.

Vehicle:

no

Details on test solutions:

- Because the test material was an UVCB (Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Materials), the test solution was prepared using the saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23. The saturated test material solution (mg/L nominal loading rate WAF) was prepared by dispersing/dissolving the needed amount of test material into the test medium (ISO Medium) two days (Day-2) before the start of the treatment.
- This solution was shaken for approximately 24 hours at approximately 30 °C (Day -2) and then was equilibrated for approximately 24 hours at approximately 20 °C (Day -1). The non-dissolved test materials were removed by filtration through a fine (0.22 μm) filter to give the 100 % saturated solution.
- As only limit test was carried out, further dilution of this saturated solution was not performed.

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:

yes

Total exposure duration:

72 h

Test temperature:

22.7 – 22.8 °C

pH:

7.25 – 8.70

Nominal and measured concentrations:

- Nominal: 100 mg/L WAF
- Measured mean concentration 72.5 mg/L
- As the measured concentration at the end of the exposure deviated more than 20 % from the measured concentration at the beginning, biological results are based on the measured mean concentration 72.5 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 control: 6

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 8335 lux (equivalent to ~112.6 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 24h, 48h and 72h) to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Range finding study test concentrations: 0.1, 1, 10 and 100 mg/L
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 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.
- Results used to determine the conditions for the definitive study: Yes, no toxicity was observed in the preliminary concentration range-finding test. All applied test concentrations (including the control) the algal cells were normal, no morphological deviations were observed. Therefore 100 mg/L test material nominal loading rate (WAF) 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 – t1) + [(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.:

> 72.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 72.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 72.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

72.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: biomass, growth rate and yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 72.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: biomass, growth rate and yield

Details on results:

MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS
- There were no morphological deviations of the algal cell during the study in any of the groups.

AVERAGE SPECIFIC GROWTH RATES
- The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the 0-72 h average specific growth rate was statistically significantly different from the untreated control value, however the difference was minor, so accordingly the No Observed Effect Concentration (NOEC) determined as 72.5 mg/L WAF.

AREAS UNDER THE GROWTH CURVES
- The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the 0-72 h areas were statistically significantly different from the untreated control value, however the difference was minor, so accordingly the No Observed Effect Concentration (NOEC) was determined as 72.5 mg/L WAF.

YIELD
- The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the 0-72 h yield was statistically significantly different from the untreated control value; however the difference was minor, so accordingly the No Observed Effect Concentration (NOEC) was determined as 72.5 mg/L WAF.

CONCLUSIONS
- In conclusion, the test material had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the test material in the test medium.

Results with reference substance (positive control):

The 72h ErC 50: 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
The 72h EyC 50: 0.54 mg/L, (95 % confidence limits: 0.50 – 0.59 mg/L)
The 72h EbC 50: 0.64 mg/L, (95 % confidence limits: 0.59 – 0.70 mg/L)
These values are within the range of laboratory ring test data (see ISO Guideline No.8692).

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 were determined from the raw data.
Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample 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 2 Sample t-Test (α = 0.05) by TOXSTAT software.

Validity

- The cell density in the control cultures increased by the factor of 69.17 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 11.93 %.

- The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.70 %.

- 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

Test group	Growth rate μ and % inhibition of μ
	0–24 h		0–48 h		0–72 h
	µ	%	µ	%	µ	%
Control	0.0573	0.0	0.0599	0.0	0.0588	0.0
72.5 mg/L WAF	0.0553+	3.5	0.0583+	2.6	0.0568*	3.4

*: Statistically significantly different compared to the control values (2 Sample 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.

 

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

Test group	Areas under the Growth Curves (A) and % inhibition of A
	0–24 h		0–48 h		0–72 h
	A	%	A	%	A	%
Control	36.0	0.0	274.0	0.0	1294.0	0.0
72.5 mg/L WAF	34.0	5.6	254.0	7.3	1146.0*	11.4

*: Statistically significantly different compared to the control values (2 Sample t-Test; α=0.05).

 

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

Test group	Yield (Y) and % inhibition of Y (0-72 h)
	Y	%
Control	68.2	0.0
72.5 mg/L WAF	58.8*	13.7

*: Statistically significantly different compared to the control values (2 Sample t-Test; α=0.05).

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study the 72 hour EbC50, ErC50 and EyC50 were >72.5 mg/L WAF. The 72 hour NOEC was 72.5 mg/L WAF and the 72 hour LOEC was >72.5 mg/L WAF. The test material had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the test material in the test medium.

Executive summary:

The toxicity of the test material to aquatic algae was investigated in accordance with the standardise guidelines OECD 201, EU Method C.3, 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.

No toxicity was observed in the preliminary concentration range-finding test, therefore 100 mg/L test material nominal loading rate (WAF) and one control was used in the main test.

The concentration of the test material was analytically determined at the start and at the end of the experiment. The measured concentration was 79.1 mg/L at the start and 65.8 mg/L at the end of the experiment. As the measured concentration at the end of the exposure deviated more than 20 % from the measured concentration at the beginning, biological results are based on the geometric mean measured concentration 72.5 mg/L WAF.

The test design included 6 replicates at test concentration and 6 replicates for the untreated control.

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 2 Sample t-Test (α = 0.05) by TOXSTAT software.

The ErC50, EbC50 and EyC50 values of the Test Item were determined directly from the raw data.

Under the conditions of this study the 72 hour EbC50, ErC50 and EyC50 were >72.5 mg/L WAF. The 72 hour NOEC was 72.5 mg/L WAF and the 72 hour LOEC was >72.5 mg/L WAF.

The test material had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the test material in the test medium.

Description of key information

Under the conditions of this study the 72 hour EbC50, ErC50 and EyC50 were >72.5 mg/L WAF. The 72 hour NOEC was 72.5 mg/L WAF and the 72 hour LOEC was >72.5 mg/L WAF. The test material had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the test material in the test medium.

Additional information

The toxicity of the test material to aquatic algae was investigated in accordance with the standardise guidelines OECD 201, EU Method C.3, 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.

No toxicity was observed in the preliminary concentration range-finding test, therefore 100 mg/L test material nominal loading rate (WAF) and one control was used in the main test.

The concentration of the test material was analytically determined at the start and at the end of the experiment. The measured concentration was 79.1 mg/L at the start and 65.8 mg/L at the end of the experiment. As the measured concentration at the end of the exposure deviated more than 20 % from the measured concentration at the beginning, biological results are based on the geometric mean measured concentration 72.5 mg/L WAF.

The test design included 6 replicates at test concentration and 6 replicates for the untreated control.

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 2 Sample t-Test (α = 0.05) by TOXSTAT software.

The ErC50, EbC50 and EyC50 values of the Test Item were determined directly from the raw data.

Under the conditions of this study the 72 hour EbC50, ErC50 and EyC50 were >72.5 mg/L WAF. The 72 hour NOEC was 72.5 mg/L WAF and the 72 hour LOEC was >72.5 mg/L WAF.

The test material had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the test material in the test medium.