2,5-dichloro-4-[[2-(dibutylamino)-4-methyl-6-[[2-(4-sulphophenyl)ethyl]amino]-5-pyrimidinyl]azo]benzenesulphonic acid, sodium salt

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:

February 27 to March 02, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28th July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For determination of the test item concentration, samples will be taken from the treatment groups and the control at the start and at the end of the test.

Vehicle:

no

Details on test solutions:

Firstly, a stock solution was prepared in RO-water. 0.5 g of test item was dispersed in 50 ml RO-water in order to obtain the concentration of 10000 mg/l. The solutions of subsequent lower concentrations were prepared by appropriate diluting of this stock solution in RO-water.
Thereafter, the appropriate amounts of these test item solutions in RO-water were diluted further in the dilution water (OECD medium) in order to obtain the test concentrations.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain number: 61.81 SAG
- Source: the algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2 Göttingen, Germany.

PRE-CULTURING
The pre-culture was prepared with OECD Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. The pre-culture was incubated for four days at this test.
The algal cultures used in the study did not contain deformed or abnormal cells.

BREEDING CONDITIONS
The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardized conditions according to the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.2 – 23.8 °C

pH:

7.90 – 9.01

Nominal and measured concentrations:

Nominal 1.024, 2.56, 6.4, 16.0, 40.0 and 100.0 mg/l
Geom. mean: 0.91, 2.42, 5.79, 15.06, 39.26 and 103.82 mg/l

Details on test conditions:

TEST SYSTEM
- Climate chamber: the test containers will be kept during the test in a climate chamber or appropriate incubator, or in a laboratory with controlled environmental conditions.
- Test vessel: 250 ml Erlenmeyer flasks.
- Type: the flasks were covered with air-permeable stoppers.
- Shake: continuous shake by a laboratory orbital shaker.
- Initial cells density: approximately 10^4 algal cells per ml test medium.
- No. of vessels per concentration: 3 replicates per test concentration.
- No. of vessels per control: 6 replicates in the control.

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: reconstituted algal growth medium (OECD medium, according to OECD 201).

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination.
- Light intensity and quality: approximately 8000 lux, which were ensured with fluorescent lamps (with a spectral range of 400-700 nm).

EFFECT PARAMETERS MEASURED
The cell concentration will be determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. Any morphological deviations of the algal cells noted will be reported.

MEASUREMENTS
- Temperaturetemperature was checked at the beginning of the study and every 24 hours in a flask filled with water. In addition, the temperature was continuously measured (with a min/max thermometer).
- Light: the light intensity was checked and recorded at the start of the test.
- pH: the pH was measured at the beginning and at the end of the study in the control and each concentration level.

TEST CONCENTRATIONS
- Exposure period: 72 hours.
- Test cocnentrations: 0.1, 1.0, 10.0 and 100 mg/l
- No. of vessels per concentration: 2 replicates in treatment groups.
- No. of vessels per control: 2 replicates in the control.
- Results used to determine the conditions for the definitive study: the percentages of growth rates inhibition were -7.05, -0.22, 20.36 and 39.83 at 0.1, 1.0, 10.0 and 100 mg/l, respectively
- Spacing factor for main test concentrations: 2.5

VALIDITY CRITERIA
- The cell concentration in the untreated control cultures should have increased by a factor of at least 16 within three days (72 hours), corresponding to a specific growth rate of 0.92 day-1.
- The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-h tests) in the control cultures must not exceed 35 %.
- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7 %.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 103.82 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.:

10.93 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Average Specific Growth Rate
The results show that the 0-72 h average specific growth rate was statistically not significantly different from the untreated control value in the concentrations of 0.91, 2.42 and 5.79 mg/l (measured) and significantly different in the 15.06, 39.26 and 103.82 mg/l concentrations (measured).
Based on the Probit analysis: the 72-h ErC10 value is determined to be 6.18 mg/l (3.801 – 8.906 mg/l); the 72-h ErC20 value is determined to be 21.79 mg/l (15.731 – 30.580 mg/l); the 72-h ErC50 value is determined to be > 103.82 mg/l (estimated: 243.01 mg/l).

Yield
The results of the statistical evaluation show that the 0-72 h yield was statistically significantly different from the untreated control value at all the tested.
Based on the Probit analysis: the 72-h EyC10 value is determined to be 0.76 mg/l (0.285 – 1.405 mg/l); the 72-h EyC20 value is determined to be 1.89 mg/l (0.932 – 3.051 mg/l); the 72-h EyC50 value is determined to be 10.93 mg/l (7.482 – 16.137 mg/l).

Morphological Changes of Algal Cells
No abnormal shape of algal cells was noticed in the control during the test.
Thin cells were noticed in the concentrations of 0.91, 2.42, 5.79, 15.06, 39.25 and 103.82 mg/l (measured) together with normal sized cells after 24, 48 and 72 hours. Thin cells, normal cells and pinches were observed in the concentration of 5.79 mg/l (measured) after 24 hours.

MEASURED CONCENTRATIONS
The measured concentration deviated by more than 20 % from the nominal at the end of the test in the test concentration of 1.024 mg/l. Therefore, the geometric means of the measured concentrations (in each group) were calculated to determine the exposure concentrations. The corresponding calculated geometric mean measured concentrations were the followings: 0.91, 2.42, 5.79, 15.06, 39.26 and 103.82 mg/l. Biological results and endpoints are based on the geometric mean measured concentrations.

VALIDITY CRITERIA
- The cell density in the control cultures increased by a factor of 133.67 within 72 hours. This corresponds to a specific growth rate of 1.62/day
- The mean coefficient of variation (CV) for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72 h-tests) in the control cultures did not exceed 35 %: CV for section-by-section growth rate day 0-1 was 55.06 %; CV for section-by-section growth rate day 1-2 was 13.80 %; CV for section-by-section growth rate day 2-3 was 9.24 %. The mean coefficient of variation for section-by-section specific growth rates: 26.04 %.
- The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 5.18 %.

Results with reference substance (positive control):

ErC50 (72h): 0.82 mg/l, (95 % confidence limits: 0.57 – 1.25 mg/l)
EyC50 (72h): 0.51 mg/l, (95 % confidence limits: 0.45 – 0.57 mg/l)

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

Concentration [mg/l]		Growth Rates (µ) and % Inhibition of µ
		0–24 h		0–48 h		0–72 h
nominal	measured	µ	%	µ	%	µ	%
Control	Not detected	0.0269	0.0	0.0693	0.0	0.0676	0.0
1.024	0.91	0.0401	-49.3	0.0711	-2.6	0.0670	0.9
2.56	2.42	0.0345	-28.4	0.0681	1.7	0.0630	6.8
6.4	5.79	0.0289	-7.4	0.0664	4.2	0.0621	8.2
16	15.06	0.0289	-7.4	0.0518	25.3	0.0550	18.7
40	39.26	0.0193	28.4	0.0483	30.2	0.0505	25.2
100	103.82	0.0193	28.4	0.0439	36.6	0.0419	38.1

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

Concentration [mg/l]		Yield (y) and % Inhibition of y
		0–72 h
nominal	measured	y	%
Control	Not detected	132.67	0.0
1.024	0.91	124.67	6.0
2.56	2.42	94.00	29.1
6.4	5.79	87.00	34.4
16	15.06	51.67	61.1
40	39.26	38.67	70.9
100	103.82	19.67	85.2

Morphological changes of algal cells

Concentration [mg/l]		Morphological changes
nominal	measured	24 h	48 h	72 h
Control	-	normal cells	normal cells	normal cells
1.024	0.91	normal cells	normal cells	normal cells
		thin cells	thin cells	thin cells
2.56	2.42	normal cells	normal cells	normal cells
		thin cells	thin cells	thin cells
6.4	5.79	normal cells	normal cells	normal cells
		thin cells	thin cells	thin cells
		pinches
16	15.06	normal cells	normal cells	normal cells
		thin cells	thin cells	thin cells
40	39.26	normal cells	normal cells	normal cells
		thin cells	thin cells	thin cells
100	103.82	normal cells	normal cells	normal cells
		thin cells	thin cells	thin cells

Calculation of exposure concentrations

Nominal conc, mg/l	Meas. conc. mg/l	% of the nominal	Meas. conc. mg/l	% of the nominal	Geom. mean [mg/l]
	0hr		72 hrs
Control	-	-		-	-
1.024	1.11	109	0.746	73	0.91
2.56	2.75	108	2.13	83	2.42
6.4	5.83	91	5.75	90	5.79
16	15.73	98	14.42	90	15.06
40	40.73	102	37.84	95	39.26
100	106.2	106	101.5	101	103.82

Results of the preliminary range-finding test

Nominal concentrations [mg/l]	Untreated  control	0.1	1	10	100
Average cell number at 72 hours (x 10^4 cells/ml)	77.5	102.5	76.5	31.5	13.5
Growth Rates (µ) [0-72h]	0.0601	0.0643	0.0602	0.0478	0.0361
% Inhibition of µ [0-72h]	–	-7.05	-0.22	20.36	39.83
Yield (Y) [0-72h]	76.5	101.5	75.50	30.50	12.50
% Inhibition of Y [0-72h]	–	-32.68	1.31	60.13	83.66

Validity criteria fulfilled:

yes

Conclusions:

ErC50 (72h) > 103.82 mg/l (geom. mean)
EyC50 (72h): 10.93 mg/l (geom. mean)

Executive summary:

The potential toxicity of the test item to aquatic algae was investigated using Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), according to the method and procedures utlined into the OECD guideline 201.

Nominal concentrations of 1.024, 2.56, 6.4, 16.0, 40.0 and 100.0 mg/l were investigated in the main study. A control was tested in parallel.

For the determination of the test item concentrations, samples were analyzed using a validated HPLC column with UV detection analytical method. The measured concentration deviated by more than 20 % from the nominal at the end of the test in the test concentration of 1.024 mg/l. Therefore, the geometric means of the measured concentrations (in each group) were calculated to determine the exposure concentrations. The corresponding calculated geometric mean measured concentrations were the followings: 0.91, 2.42, 5.79, 15.06, 39.26 and 103.82 mg/l Biological results and endpoints are based on the geometric mean measured concentrations.

Exponentially-growing cultures of Raphidocelis subcapitata were exposed to the test item under defined conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included three replicates at each test concentration and six replicates for the untreated control.

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

Conclusion

ErC50 (72h) > 103.82 mg/l meas (geom. mean)

EyC50 (72h): 10.93 mg/l meas. (geom. mean)

Description of key information

Not harmful/toxic to aquatic algae and cyanobacteria

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

6.18 mg/L

Additional information

The potential toxicity of the test item to aquatic algae was investigated using Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), according to the method and procedures utlined into the OECD guideline 201.

Nominal concentrations of 1.024, 2.56, 6.4, 16.0, 40.0 and 100.0 mg/l were investigated in the main study. A control was tested in parallel.

For the determination of the test item concentrations, samples were analyzed using a validated HPLC column with UV detection analytical method. The measured concentration deviated by more than 20 % from the nominal at the end of the test in the test concentration of 1.024 mg/l. Therefore, the geometric means of the measured concentrations (in each group) were calculated to determine the exposure concentrations. The corresponding calculated geometric mean measured concentrations were the followings: 0.91, 2.42, 5.79, 15.06, 39.26 and 103.82 mg/l Biological results and endpoints are based on the geometric mean measured concentrations.

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

The results show that the 0-72 h average specific growth rate was statistically not significantly different from the untreated control value in the concentrations of 0.91, 2.42  and 5.79 mg/l (measured) and significantly different in the 15.06, 39.26 and 103.82 mg/l concentrations (measured). Based on the Probit analysis: the 72-h ErC10 value is determined to be 6.18 mg/l (3.801 – 8.906 mg/l); the 72-h ErC20 value is determined to be 21.79 mg/l (15.731 – 30.580 mg/l); the 72-h ErC50 value is determined to be > 103.82 mg/l (estimated: 243.01 mg/l). On the basis of the Dunnett t-test statistical evaluation, the 72-h NOEC based on growth rate is determined to be 5.79 mg/l and the the 72-h LOEC based on growth rate is determined to be 15.06 mg/l.

The results of the statistical evaluation show that the 0-72 h yield was statistically significantly different from the untreated control value at all the tested.

Based on the Probit analysis: the 72-h EyC10 value is determined to be 0.76 mg/l (0.285 – 1.405 mg/l); the 72-h EyC20 value is determined to be 1.89 mg/l (0.932 – 3.051 mg/l); the 72-h EyC50 value is determined to be 10.93 mg/l (7.482 – 16.137 mg/l).

No abnormal shape of algal cells was noticed in the control during the test. Thin cells were noticed in the concentrations of 0.91, 2.42, 5.79, 15.06, 39.25 and 103.82 mg/l (measured) together with normal sized cells after 24, 48 and 72 hours. Thin cells, normal cells and pinches were observed in the concentration of 5.79 mg/l (measured) after 24 hours.