2-[[4-chloro-6-[[8-hydroxy-3,6-disulpho-7-[(1-sulpho-2-naphthyl)azo]-1-naphthyl]amino]-1,3,5-triazin-2-yl]amino]-5-sulphobenzoic 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:

13 October 2017 to 16 October 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)

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:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 2-7-3. 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:

Analytical measurement was performed at the control and at the applied test concentration levels at the beginning and at the end of the experiment.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Test Material
A stock solution with a concentration of 100 mg/L was prepared with the test material and algal growth medium (OECD Medium). The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae.

- Untreated Control
Algal growth medium (OECD medium) was inoculated with algal cells (without test material) and was examined in parallel to the test material concentrations.

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.

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:

22.6 - 22.7°C (flask); 21.8 - 23.2°C (climate chamber)

pH:

7.22 - 8.42

Nominal and measured concentrations:

6.25, 12.5, 25.0, 50.0 and 100.0 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Aeration: no. Flasks were continuously shaken by a laboratory orbital shaker
- No. of vessels per concentration: 3 replicates per treated and light filter group at each concentration level
- No. of vessels per vehicle control: 6

PERFORMANCE OF THE TEST
The test material was a water soluble deep-coloured powder. A spectral analysis of appropriate concentrations was made, to show the absorption of light at the wavelengths required by chlorophyll. As the amount of light for photosynthesis was likely to be significantly affected by the shading effect of the coloured test solution, therefore a modified test was performed in order to separate physical light absorption effect of the coloured material from any systemic toxic effects.
To assure the appropriate position of the test solutions, two superposed flat flasks (which are open for the light only from above) were used per replicate for this experiment.
The following three types of treatment was tested: control, treated and light filter groups. In each case, the algal cells were in the lower flask, where the only light available passes through the upper flat flask (the sides of the flask was covered). The depth of the liquid in the upper flask was 50% of that in the lower flask (on the basis that the ‘average’ algal cell in the lower flask were suspended at the point of 50% of the depth).
The flasks were continuously shaken by a laboratory orbital shaker during the exposure in order to keep the alga cells in suspension. Volume of algal suspension in the lower flask will be approximately 20 mL per replicate.

GROWTH MEDIUM
- Standard medium used: yes - algal growth medium (OECD Medium)

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.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: The light intensity at the position occupied by algal culture flasks during the test was about 8373 lux (equivalent to ~113 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 (in each group) and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.
The light absorptions of the control and the Test Item solutions were measured photometrically to show the absorption of light at the wavelengths required by chlorophyll. The light adsorption measurements were performed in the range of 350-700 nm because the absorption maximum of the Chlorophyll A is about 430 and 660 nm and the absorption maximum of Chlorophyll B is about 450 and 640 nm.


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.1, 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: yes
Algal cells were exposed to each concentration of the test material plus a control, for 72 hours. The test was performed with 2 replicates per each test concentration and 3 replicates in the control group.
Because toxic response was observed during the preliminary range-finding test, five test concentrations in a geometric series with a separation factor of 3.2 and one control were used in the main experiment. The reduced growth could be caused by light absorption by the test material, or by a direct toxic effect.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

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

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS
There was no morphological deviation observed in both of the treated and filter group in any of the investigated concentration levels during the whole experiment.

LIGHT ABSORPTION MEASUREMENTS
The light absorptions of the control and the test material solutions were measured photometrically in the range of 350-700 nm. The absorption maximum of the Chlorophyll A is about 430 and 660 nm and the absorption maximum of Chlorophyll B is about 450 and 640 nm.
The maximum light absorption of the test material was at approximately at 546 nm in the test solutions. However, there was significant absorption at wavelengths required by chlorophyll in the test solutions as well.

GROWTH INHIBITION
The results of this experiment showed a clear inhibition effect, particularly at higher concentrations which, was attributed to the light absorption by the test material. However, inhibition was also attributed the test material being in contact with the algae cells. At lower concentrations (12.5 and 25 mg/L) the inhibition attributed to direct contact was greater than attributed to light absorption. This indicates that there was also a direct toxic effect on the growth of the alga (Pseudokirchneriella subcapitata) although the EC50 was considered to be >100 mg/L.

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 corrected EbC50 and EyC50 values of the test material were determined directly from the raw data. The corrected ErC50, the non-corrected ErC50, EbC50 and EyC50 values of the test material and their confidence limits were calculated using Probit analysis by TOXSTAT software.
Statistical comparisons of biomass, average specific growth rates and yield in control and in test material groups (filter and treated groups) were carried out using analysis of variance (ANOVA), Bonferroni t-Test and Dunnett’s 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 and Dunnett’s Test.
The corrected EbC50, EyC50 and ErC50 values of the test material (treated minus filter, if applicable) and their confidence limits will be calculated using Probit analysis by TOXSTAT software or will be determined directly from the raw data.

Table 1: Growth Rates (µ) during the Test Period

Nominal conc (mg/L)		Growth rate (µ)			Difference (treated-filtered)
		0-24 h	0-48 h	0-72 h
Control		0.0593	0.0615+	0.0590	0.0
6.25	treated	0.0498	0.0594	0.0583*	0.0005
	filter	0.0498	0.0597	0.0587
12.5	treated	0.0401+*	0.0559*	0.0519*	0.0062*
	filter	0.0498	0.0567	0.0581+
25	treated	0.0401+*	0.0512*	0.0500+*	0.0082*
	filter	0.0498	0.0568	0.0582
50	treated	0.0345*	0.0437*	0.0439*	0.0081*
	filter	0.0458*	0.0538*	0.0520*
100	treated	0.0345*	0.0373*	0.0378*	0.0139*
	filter	0.0458*	0.0545*	0.0517*

*   : 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.

Table 2: Percentage inhibition of 72h Growth Rates (µ)

Nominal conc (mg/L)		% inhibaition of µ (0-72h)
		not corrected % inhibition	corrected % inhibition (treated-filtered)
Control		-	-
6.25	treated	1.3	0.8
	filter	0.5
12.5	treated	12.1	10.5
	filter	1.6
25	treated	15.4	13.9
	filter	1.4
50	treated	25.7	13.8
	filter	11.9
100	treated	36	23.5
	filter	12.4

 

Table 3: Area under the Growth Curves (A) during the Test Period

Nominal conc (mg/L)		Area under the growth curves (A)			Difference (treated-filtered)
		0-24 h	0-48 h	0-72 h
Control		38	294	1342
6.25	treated	28	252*	1232	36
	filter	28	256	1268
12.5	treated	20*	204*	860*	316*
	filter	28	228*	1176*
25	treated	20*	168*	724*	456*
	filter	28	228*	1180*
50	treated	16*	120*	480*	306*
	filter	24*	196*	840*
100	treated	16*	92*	324*	512*
	filter	24*	200*	836*

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

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the 72 hour ECr50 was determined to be in excess of 100 mg/L. The 72 hour NOECr was determined to be 6.25 mg/L.

Executive summary:

The effect of the test material was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 201, EU Method C.3, US EPA OCSPP 850.5400 and JMAFF 2-7-3.

The test material was a water soluble deep-coloured powder. A spectral analysis of appropriate concentrations was made and reported, to show the absorption of light at the wavelengths required by chlorophyll. As the amount of light for photosynthesis was likely to be significantly affected by the shading effect of the coloured test solution, therefore a modified test was performed in order to separate physical light absorption effect of the coloured material from any systemic toxic effects. Modified test results and results without taking into account modification are reported separately (according to OECD 201).

Five test concentrations in a geometric serieswith a separation factor of 2.0 and one untreated control were tested in the main experiment. The test design included 3 replicates at each test group (light filter and treated group) and 6 replicates for the untreated control.

The nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25, 50 and 100 mg/L. As the measured concentrations deviated not more than 20% from the nominal, biological results are based on the nominal concentrations.

Statistical comparisons of biomass, average specific growth rates and yield in control and in test material groups (filter, treated groups and the subsequent corrected values) were carried out using analysis of variance (ANOVA), Bonferroni t-Test by TOXSTAT software.

The corrected EbC50, ErC5 0and EyC50 values of the test material were determined directly from the raw data. The non-corrected EbC50 and EyC50 values of the test material and their confidence limits were calculated using Probit analysisby TOXSTAT software.

Under the conditions of the study the 72 hour ECr50 was determined to be in excess of 100 mg/L. The 72 hour NOECr was determined to be 6.25 mg/L.

The results of this experiment showed a clear inhibition effect, particularly at higher concentrations which, was attributed to the light absorption by the test material. However, inhibition was also attributed the test material being in contact with the algae cells. At lower concentrations (12.5 and 25 mg/L) the inhibition attributed to direct contact was greater than attributed to light absorption. This indicates that there was also a direct toxic effecton the growth of the alga (Pseudokirchneriella subcapitata) although the EC50 was considered to be >100 mg/L.

Description of key information

Under the conditions of the study the 72 hour ECr50 was determined to be in excess of 100 mg/L. The 72 hour NOECr was determined to be 6.25 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

6.25 mg/L

Additional information

The effect of the test material was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 201, EU Method C.3, US EPA OCSPP 850.5400 and JMAFF 2-7-3. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was a water soluble deep-coloured powder. A spectral analysis of appropriate concentrations was made and reported, to show the absorption of light at the wavelengths required by chlorophyll. As the amount of light for photosynthesis was likely to be significantly affected by the shading effect of the coloured test solution, therefore a modified test was performed in order to separate physical light absorption effect of the coloured material from any systemic toxic effects. Modified test results and results without taking into account modification are reported separately (according to OECD 201).

Five test concentrations in a geometric serieswith a separation factor of 2.0 and one untreated control were tested in the main experiment. The test design included 3 replicates at each test group (light filter and treated group) and 6 replicates for the untreated control.

The nominal concentrations of test material used in the main experiment were: 6.25, 12.5, 25, 50 and 100 mg/L. As the measured concentrations deviated not more than 20% from the nominal, biological results are based on the nominal concentrations.

Statistical comparisons of biomass, average specific growth rates and yield in control and in test material groups (filter, treated groups and the subsequent corrected values) were carried out using analysis of variance (ANOVA), Bonferroni t-Test by TOXSTAT software.

The corrected EbC50, ErC5 0and EyC50 values of the test material were determined directly from the raw data. The non-corrected EbC50 and EyC50 values of the test material and their confidence limits were calculated using Probit analysisby TOXSTAT software.

Under the conditions of the study the 72 hour ECr50 was determined to be in excess of 100 mg/L. The 72 hour NOECr was determined to be 6.25 mg/L.

The results of this experiment showed a clear inhibition effect, particularly at higher concentrations which, was attributed to the light absorption by the test material. However, inhibition was also attributed the test material being in contact with the algae cells. At lower concentrations (12.5 and 25 mg/L) the inhibition attributed to direct contact was greater than attributed to light absorption. This indicates that there was also a direct toxic effecton the growth of the alga (Pseudokirchneriella subcapitata) although the EC50 was considered to be >100 mg/L.