Reaction products of 1-(substitutedphenyl)urea coupled with diazotated potassium sodium substituted-5-{[2-(substituted)ethyl]sulfonyl}benzenesulfonate, further condensed with 2,4,6-trichloro-1,3,5-triazine, further converted with disubstituted benzene-1,4-disulfonic acid in aq. sodium hydroxide

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:

The study was conducted between 12 October 2015 and 19 November 2015

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
Only samples at the No Observed Effect Concentration (NOEC) were analyzed.

Vehicle:

no

Details on test solutions:

Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

RANGE_FINDING TEST:
The test concentrations to be used in the definitive test were determined by preliminary range-finding tests. The initial range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours.

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. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium.

A nominal amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (2.5 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

The control group was maintained under identical conditions but not exposed to the test item.

Given that the test item formed colored test solutions, spectrophotometer readings were taken at both 460 and 665 nm in order to determine whether significant light absorption occurred.

At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 ºC 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.

After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.

The results of the initial range-finding test showed inhibition of yield occurred, particularly at 100 mg/L where significant light absorption was observed and so a second range-finding test was conducted in accordance with the recommendations of the OECD Guidance Document No. 23 on Aquatic Testing of Difficult Substances and Mixtures for the testing of colored substances.

The test was conducted in 250 mL glass conical flasks each containing 25 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium.

A nominal amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (200 mL) of each of the stock solutions was separately inoculated with algal suspension (1.2 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

The control group was maintained under identical conditions but not exposed to the test item.

At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 10000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

DEFINITIVE TEST
Based on the results of the range-finding tests and at the request of the Sponsor, the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

Experimental Preparation
A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 32, 10, 3.2 and 1.0 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (3.3 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.

Exposure Conditions
As in the second range-finding test 250 mL glass conical flasks were used. Six flasks each containing 25 mL of test preparation were used for the control and three flasks each containing 25 mL were used for each treatment group.

The control group was maintained under identical conditions but not exposed to the test item.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.

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 21 ± 1 degree C.

Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 degree C until the algal cell density was approximately 104 - 105 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Not available

Test temperature:

24ºC ± 1

pH:

7.6 to 7.7

Dissolved oxygen:

Not available

Salinity:

Not applicable

Conductivity:

Not available

Nominal and measured concentrations:

Nominal concentrations: 1.0, 3.2, 10, 32 and 100 mg/L
Analysis of the 100 mg/L test preparations at 0 and 72 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type (delete if not applicable): closed, plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: 25mL
The test was conducted using ASTM culture medium with no media renewal (static test).
- Initial cells density: of 5E+03 cells per mL
- Control end cells density: 8.23E+05 cells per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
The culture medium was prepared using reverse osmosis purified deionized water.

OTHER TEST CONDITIONS
- Adjustment of pH: the pH was adjusted to 7.5 with 0.1N NaOH or HCl.
- Photoperiod: constant illumination
- Light intensity and quality: intensity approximately 10000 lux provided by warm white lighting (380 – 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter®Multisizer Particle Counter.

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

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

RESULTS:
RANGE-FINDING TESTS:
The results of the initial range-finding test showed effects on growth rate at all test concentrations. The results of the second range finding test showed no effect on growth rate at the test concentration of 0.10/1.0 and 10 mg/L. However, inhibition of yield was observed at 10 and 100 mg/L.
Based on this information, and at the request of the sponsor test concentration of 1.0/3.2/10/32 and 100 mg/L were selected for the definitive test.
Chemical analysis of the 1.0/10 and 100 mg/L test preparations taken from the initial range-finding test at 0 and 72 hours showed measured concentrations to range from 95% to 104% of nominal indicating that the test item was stabe under test conditions.

DEFINITIVE TEST:
Verification of test concentration:
Analysis of the 100 mg/L test preparations at 0 and 72 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

GROWTH DATA:
From the data obtained, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were moderatly affected by the presence of the test item over the 72-hour exposure period.

Accordingly of growth Rate:
ErC10 (0-72 h) : > 100 mg/L
ErC20 (0-72 h) : > 100 mg/L
ErC50 (0-72 h) : > 100 mg/L

Where ErCx is the test concentratio that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control all test concentrations using Welch's t-test with Bonferroni correction. There were no statistically significant differences (P>= 0.05), between the control and all test groups and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100 mg/L.

Inhibition of Yield:
EyC10 (0-72 h) : 28 mg/L
EyC20 (0-72 h) : > 100 mg/L
EyC50 (0-72 h) : > 100 mg/L

Where:
EyCx is the test concentration that reduced yield by x%.
There were no statistically significant differences (P>= 0.05), between the control and all test groups and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 100 mg/L.

Results with reference substance (positive control):

Positive Control
A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. The positive control was conducted in a separate study (Study Number 41501180) between 12 May 2015 and 15 May 2015.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Results with reference substance valid? Yes
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.0 mg/L; 95% confidence limits 0.90 – 1.2 mg/L
EyC50 (0 – 72 h) : 0.49 mg/L; 95% confidence limits 0.42 – 0.58 mg/L

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

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Observations on 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.

Observations on Test Item Solubility

At the start of the test all control cultures were observed to be clear colorless solutions. The test cultures were observed to range from extremely pale yellow solutions at 1.0 mg/L through to dark yellow solutions at 100 mg/L. After the 72-Hour test period all control cultures were observed to be green dispersions. The test cultures were observed to range from pale yellow/green dispersions at 1.0 mg/L through to dark yellow solutions at 100 mg/L.

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 165 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:5.00 x 103 cells per mL

- Mean cell density of control at 72 hours:8.23 x 105 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 27% 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 (0-72h) was 2% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Analytical analysis result of the test item concentrations

Results for Range-Finding Samples

Time point	Nominal Concentration of Test Item in Range-Finding Sample cnom	Sample Preparation Factor F	Determined Concentration of Test Item in Range-Finding Sample c	% of Nominal Concentration
[hours]	[mg/L]		[mg/L]	[%]
0	1.0	1	1.01	101
	10	1	10.4	104
	100	1	103	103
72	1.0	1	0.952	95
	10	1	10.2	102
	100	1	103	103

Results for Test Samples

Time point	Nominal Concentration of Test Item in Test Sample cnom	Sample Preparation Factor F	Determined Concentration of Test Item in Test Sample   c	% of Nominal Concentration
[hours]	[mg/L]		[mg/L]	[%]
0	Control	1	<LOQ	-
	100	1	90.5	90
72	Control	1	<LOQ	-
	100	1	92.9	93

 

LOQ =             Limit of Quantification

-      =             not applicable

Cell densities and percentage inhibition of growth

Initial Range-finding Test

Nominal Concentration (mg/L)		Cell Densities*(cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	5.89E+03	7.81E+05	-	-
	R2	5.17E+03	8.00E+05
	Mean	5.53E+03	7.91E+05
0.10	R1	6.16E+03	6.28E+05	6	16
	R2	6.45E+03	7.10E+05
	Mean	6.30E+03	6.69E+05
1.0	R1	5.81E+03	6.87E+05	7	27
	R2	5.95E+03	4.73E+05
	Mean	5.88E+03	5.80E+05
10	R1	5.38E+03	4.01E+05	13	44
	R2	6.34E+03	4.95E+05
	Mean	5.86E+03	4.48E+05
100	R1	5.52E+03	3.14E+05	17	57
	R2	6.08E+03	3.80E+05
	Mean	5.80E+03	3.47E+05

 

Second Range-finding Test

Nominal Concentration (mg/L)		Cell Densities* (cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	4.22E+03	2.60E+05	-	-
	R2	3.29E+03	4.42E+05
	Mean	3.75E+03	3.51E+05
0.10	R1	4.46E+03	5.60E+05	[8]	[57]
	R2	3.78E+03	5.41E+05
	Mean	4.12E+03	5.51E+05
1.0	R1	3.67E+03	4.58E+05	[2]	[32]
	R2	5.51E+03	4.65E+05
	Mean	4.59E+03	4.62E+05
10	R1	4.08E+03	3.55E+05	3	12
	R2	3.46E+03	2.66E+05
	Mean	3.77E+03	3.10E+05
100	R1	4.96E+03	1.70E+05	21	55
	R2	3.90E+03	1.54E+05
	Mean	4.43E+03	1.62E+05

*    Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R₁and R₂= Replicates 1 and 2

Definitive Test

Nominal Concentration (mg/L)		pH	Cell Densities* (cells per mL)			pH
		0 h	24 h	48 h	72 h	72 h
Control	R1	7.6	1.69E+04	1.14E+05	8.14E+05	7.7
	R2		1.57E+04	9.79E+04	8.31E+05
	R3		1.77E+04	9.38E+04	6.83E+05
	R4		1.53E+04	1.03E+05	9.58E+05
	R5		2.40E+04	1.07E+05	8.73E+05
	R6		1.45E+04	1.08E+05	7.78E+05
	Mean		1.73E+04	1.04E+05	8.23E+05
1.0	R1	7.7	3.86E+04	2.40E+05	1.36E+06	7.7
	R2		2.11E+04	2.18E+05	1.09E+06
	R3		2.05E+04	1.90E+05	1.07E+06
	Mean		2.68E+04	2.16E+05	1.17E+06
3.2	R1	7.7	1.75E+04	1.48E+05	1.14E+06	7.7
	R2		1.73E+04	1.22E+05	1.02E+06
	R3		1.90E+04	1.34E+05	1.05E+06
	Mean		1.80E+04	1.35E+05	1.07E+06
10	R1	7.7	1.44E+04	1.18E+05	9.12E+05	7.7
	R2		1.45E+04	9.66E+04	7.43E+05
	R3		1.59E+04	1.73E+05	9.23E+05
	Mean		1.50E+04	1.29E+05	8.59E+05
32	R1	7.6	1.80E+04	1.10E+05	7.88E+05	7.7
	R2		2.38E+04	1.05E+05	6.36E+05
	R3		1.38E+04	9.86E+04	6.39E+05
	Mean		1.85E+04	1.05E+05	6.88E+05
100	R1	7.6	1.37E+04	9.29E+04	5.96E+05	7.7
	R2		1.92E+04	1.97E+05	6.49E+05
	R3		2.37E+04	2.85E+05	9.73E+05
	Mean		1.89E+04	1.92E+05	7.40E+05

*    Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R₁- R₆= Replicates 1 to 6

Daily Specific Growth Rates for the Control Cultures in the Definitive Test

		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.051	0.080	0.082
	R2	0.048	0.076	0.089
	R3	0.053	0.069	0.083
	R4	0.047	0.079	0.093
	R5	0.065	0.062	0.088
	R6	0.044	0.083	0.082
	Mean	0.051	0.075	0.086

R₁- R₆= Replicates 1 to 6

Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.071		8.09E+05
	R2	0.071		8.26E+05
	R3	0.068		6.78E+05
	R4	0.073	-	9.53E+05	-
	R5	0.072		8.68E+05
	R6	0.070		7.73E+05
	Mean	0.071		8.18E+05
	SD	0.002		9.22E+04
1.0	R1	0.078	[10]	1.35E+06
	R2	0.075	[6]	1.09E+06
	R3	0.075	[5]	1.07E+06
	Mean	0.076	[7]	1.17E+06	[43]
	SD	0.002		1.60E+05
3.2	R1	0.075	[6]	1.13E+06
	R2	0.074	[4]	1.02E+06
	R3	0.074	[5]	1.04E+06
	Mean	0.075	[5]	1.06E+06	[30]
	SD	0.001		6.05E+04
10	R1	0.072	[2]	9.07E+05
	R2	0.069	2	7.38E+05
	R3	0.072	[2]	9.18E+05
	Mean	0.071	[1]	8.54E+05	[4]
	SD	0.002		1.01E+05
32	R1	0.070	1	7.83E+05
	R2	0.067	5	6.31E+05
	R3	0.067	5	6.34E+05
	Mean	0.068	4	6.83E+05	17
	SD	0.002		8.67E+04
100	R1	0.066	6	5.91E+05
	R2	0.068	5	6.44E+05
	R3	0.073	[3]	9.68E+05
	Mean	0.069	2	7.35E+05	10
	SD	0.004		2.04E+05

*In accordance with the OECD test guideline only thean value for yield for each test concentration is calculated

R₁-R₆= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

Validation Criteria

The following data show that the cell concentration of the control culturesincreasedby a factor of 165 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                      :   5.00 x 10³cells per mL

Mean cell density of control at 72 hours                    :   8.23 x 10⁵cells per mL

 

The mean coefficient of variation for section by section specific growth rate for the control cultures was 27% 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) was2% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EC50 values of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

Executive summary:

Alga growth inhibition test.

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata according to OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Following preliminary range-finding tests,Pseudokirchneriella subcapitatawas exposed to an aqueous solution of the test item at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test was conducted using ASTM culture medium with no media renewal (static test). Given the colored nature of the test item, testing was conducted in accordance with the OECD Guidance Document No. 23 on Aquatic Testing of Difficult Substances and Mixtures whereby the light path was shortened and the light intensity increased to overcome any possible shading effects.

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

Analysis of the 100 mg/L test preparations at 0 and 72 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

Exposure of Pseudokirchneriella subcapitatato the test item gave EC₅₀values of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

Description of key information

The study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriellasubcapitata.

Following preliminary range-finding tests,Pseudokirchneriella subcapitatawas exposed to an aqueous solution of the test item at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L for 72 hours, under constant illumination and static conditions and shaking at a temperature of 24 ± 1 °C. 

Analysis of the 100 mg/L test preparations at 0 and 72 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC₅₀values of greater than 100 mg/L. 

The No Observed Effect Concentration was 100 mg/L.

It can be conclude that the test item is not toxic and have no effect on the growth of the green alga.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L