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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 12 October 2015 and 03 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Analytical monitoring:

yes

Details on sampling:

Verification of Test Concentrations
Water samples were taken from the control and 100 mg/L test vessel at 0 and 72 hours from fresh media and at 24 and 96 hours from old media for quantitative analysis. The samples were stored frozen prior to analysis.

Duplicate samples and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media) were taken and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

Range-finding Test
A nominal amount of test item (2500 mg) was dissolved in test water and the volume adjusted to 1 liter to give the 2500 mg/L stock solution from which a series of dilutions was made to give the required test concentrations of 1.0, 10, and 100 mg/L.

The stock solution was inverted several times and the prepared test solution was mixed with a flat bladed stirrer for one minute to ensure adequate mixing and homogeneity.

Definitive Test
An initial stock solution of 2500 mg/L was prepared by dissolving 2500 mg of test item into a final volume of 1 liter of diluent from which an 800 mL aliquot was added to a final volume of
20 liters of test media to give the 100 mg/L test concentration.

The stock solution was inverted several times and the prepared test solution was mixed with a flat bladed stirrer for one minute to ensure adequate mixing and homogeneity.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Test Species
The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in house since 07 October 2015. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 19 October 2015 to 26 October 2015. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

The water temperature was controlled at approximately 14 °C with a dissolved oxygen content of greater than or equal to 9.2 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test. There were no mortalities in the 7 days prior to the start of the test and the fish had a mean standard length of 3.8 cm (sd = 0.4) and a mean weight of 0.78 g (sd = 0.29) at the end of the definitive test. Based on the mean weight value this gave a loading rate of
0.27 g bodyweight/liter.
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

N/A

Test temperature:

The water temperature was recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. The temperature was measured using a Hanna Instruments HI 93510 digital thermometer. Temperature was maintained at 13 °C to 14 °C throughout the test.

pH:

The pH was recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. The pH was measured using a Hach Flexi handheld meter. There were no treatment related differences for pH. pH range: 7.6 - 8.2.

Dissolved oxygen:

The dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours, represent those of the freshly prepared test preparations while the measurements taken prior to each test media renewal, and on termination of the test after 96 hours, represent those of the used or 24-Hour old test preparations. The dissolved oxygen concentration were measured using a Hach Flexi handheld meter. There were no treatment related differences for oxygen concentration. Dissolved oxygen range: 9.1 mg/L - 10 mg/L.

Salinity:

Not applicable as freshwater was used.

Nominal and measured concentrations:

Range-finding Test: Nominal test concentrations of 1.0, 10, and 100 mg/L
Definitive Test: Nominal test concentration of 100 mg/L

Details on test conditions:

Test Water
The test water used for both the range-finding and definitive tests was the same as that used to maintain the stock fish.

Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

Procedure
Range-finding Test
The test concentration to be used in the definitive test was determined by a preliminary range-finding test.

In the range-finding test fish were exposed to a series of nominal test concentrations of 1.0, 10, and 100 mg/L. The test item was dissolved directly in test water.

A nominal amount of test item (2500 mg) was dissolved in test water and the volume adjusted to 1 liter to give the 2500 mg/L stock solution from which a series of dilutions was made to give the required test concentrations of 1.0, 10, and 100 mg/L.

The stock solution was inverted several times and each test solution was mixed with a flat bladed stirrer for one minute to ensure adequate mixing and homogeneity.

In the range-finding test three fish were placed in each test and control vessel and maintained in a temperature controlled room at approximately 14 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours under static test conditions. Each 25-30 liter test and control vessel contained 20 L of test media and was covered to reduce evaporation. After 3, 6, 24, 48, 72 and 96 hours any mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish.

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

A sample of each test concentration was taken for chemical analysis at 0 and 24 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.


Definitive Test
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines, no mortalities or sub-lethal effects of exposure were observed.


Experimental Preparation
An initial stock solution of 2500 mg/L was prepared by dissolving 2500 mg of test item into a final volume of 1 liter of diluent from which an 800 mL aliquot was added to a final volume of 20 liters of test media to give the 100 mg/L test concentration.

The stock solution was inverted several times and the prepared test solution was mixed with a flat bladed stirrer for one minute to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparation were verified by chemical analysis at 0, 24, 72 and 96 hours.


Exposure Conditions
As in the range-finding test, 25-30 liter glass exposure vessels containing 20 liters of test media were used for each control and test concentration. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at 13 to 14 °C in a temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.

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

A semi-static test regime was employed in the test involving a daily renewal of the test preparations to prevent the build up of nitrogenous waste products.

Evaluations
Test Organism Observations
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours after the start of exposure. The criteria of death were taken to be the absence of both respiratory movement and response to physical stimulation.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Range-finding Test
The results showed no mortalities at the test concentrations of 1.0 and 100 mg/L. However, mortalities were observed at 10 mg/L. The mortalities at 10 mg/L were believed to have resulted from aggressive behavior as no dose response was observed.

Chemical analysis of the test preparations at 0 and 24 hours showed that near nominal concentrations were achieved indicating that the test item was stable under test conditions.

Based on this information, a single test concentration of 100 mg/L was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the maximum test concentration given in the OECD/EC Test Guidelines, no mortalities or sub lethal effects of exposure were observed.

Definitive Test
Mortality Data
There were no mortalities in 7 fish exposed to a test concentration of 100 mg/L for a period of 96 hours.

The Lowest Observed Effect Concentration (LOEC) was considered to be greater than 100 mg/L and the No Observed Effect Concentration (NOEC) was 100 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.


Sub-Lethal Effects
There were no sub-lethal effects of exposure observed in 7 fish exposed to a test concentration of 100 mg/L for a period of 96 hours.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

An estimate of the LC50 values was given by inspection of the mortality data.

Sublethal observations / clinical signs:

Verification of Test Concentrations

Analysis of the test preparations at 0, 24, 72 and 96 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

Mortality Data

Inspection of the mortality data gave the following results:

 

Time (h)	LC50(mg/L)
3	>100
6	>100
24	>100
48	>100
72	>100
96	>100

Validation Criteria

The test was considered to be valid given that none of the control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was greater than or equal to 60% of ASV (equivalent to 6.1 mg O₂/L) in the control and test vessels.

Water Quality Criteria

The temperature was maintened at 13 °C to 14°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Observations on Test Item Solubility

Throughout the duration of the test, the test preparations were observed to be clear dark orange colored solutions.

Cumulative Mortality Data in the Range-finding Test

Nominal Concentration (mg/L)	Cumulative Mortality (Initial Population = 3)
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours
Control	0	0	0	0	0	0
1.0	0	0	0	0	0	0
10	0	0	2*	2	2	2
100	0	0	0	0	0	0

* 1 fish observed with loss of equilibrium at approximately 22 hours and 1 fish observed with damage to tail and lethargic at approximately 24 hours and humanely killed at these times, considered as mortalities at 24 hours.

Sub-lethal Effects of Exposure in the Range-finding Test

Nominal Concentration (mg/L)	Sub-lethal Effects	Time (Hours)
		3	6	24	48	72	96
Control	No abnormalities detected	3	3	3	3	3	3
1.0	No abnormalities detected	3	3	3	3	3	3
10	No abnormalities detected	3	3	1	1	1	1
	Loss of equilibrium	0	0	1^*	0	0	0
	Damage to tail and lethargic	0	0	1^**	0	0	0
100	No abnormalities detected	3	3	3	3	3	3

^* 1 fish observed with loss of equilibrium at approximately 22 hours and humanely killed at this time, considered as mortalities at 24 hours.

^** 1 fish observed with damage to tail and lethargic at approximately 24 hours and humanely killed at this time, considered as mortalities at 24 hours.

Cumulative Mortality Data in the Definitive Test

Nominal Concentration (mg/L)	Cumulative Mortality (Initial Population = 7)						% Mortality
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours	96 Hours
Control	0	0	0	0	0	0	0
100	0	0	0	0	0	0	0

Water Quality Measurements

Nominal Concentration (mg/L)	Time (Hours)
	0 Hours (Fresh Media)			24 Hours (Old Media)
	pH	mg O2/L	T ºC	pH	mg O2/L	T°C
Control	7.7	9.6	13	8.0	10.0	14
100	7.6	9.4	13	7.8	9.3	14

 

Nominal Concentration (mg/L)	Time (Hours)
	24 Hours (Fresh Media)			48 Hours (Old Media)
	pH	mg O2/L	T ºC	pH	mg O2/L	T°C
Control	7.7	9.5	13	7.6	9.5	14
100	7.6	9.4	13	7.7	9.3	14

 

Nominal Concentration (mg/L)	Time (Hours)
	48 Hours (Fresh Media)			72 Hours (Old Media)
	pH	mg O2/L	T ºC	pH	mg O2/L	T°C
Control	7.6	9.2	13	8.2	9.8	14
100	7.6	9.3	14	7.8	9.3	14

 

Nominal Concentration (mg/L)	Time (Hours)
	72 Hours (Fresh Media)			96 Hours (Old Media)
	pH	mg O2/L	T ºC	pH	mg O2/L	T°C
Control	7.7	9.1	13	7.9	9.5	14
100	7.6	9.1	13	7.9	9.4	14

 

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and based on nominal test concentrations gave a 96-Hour LC50 of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

 

Methods…….

Following a preliminary range-finding test, seven fish were exposed to an aqueous solution of the test item, at a single concentration of 100 mg/L for a period of 96 hours at a temperature of 13 to 14 °C under semi-static conditions. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

 

Results…….

Analysis of the test preparations at 0, 24, 72 and 96 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

Exposure ofrainbow trout (Oncorhynchus mykiss) to the test item gave LC₅₀values based on the nominal test concentrations of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Description of key information

The study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss).

Seven fish were exposed to an aqueous solution of FAT 40871/A TE at a single concentration of 100 mg/L for a period of 96 hours under semi-static test conditions.

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave LC50 values based on the nominal test concentrations of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

No toxicity was observed on Rainbow trout with FAT 40871/A TE.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L