Triazine derivative

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The range-finding test was conducted between 16 April 2007 and 20 April 2007 and the definitive test between 30 April 2007 and 4 May 2007.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Sponsor's identification: C-Y9
Description : yellow powder
Lot Number : MB-1
Date received : 6 March 2007
Storage conditions: room temperature in the dark over silica gel

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
100 mg/l

- Sampling method:
Water samples were taken from the control and each replicate test vessel at 0 (fresh media), 24 and 96 hours (old media) for quantitative analysis.

Duplicate samples and samples at 24 (fresh media), 48 and 72 hours (fresh and old media) were taken and stored at approximately -20°C for further analysis if necessary.

- Sample storage conditions before analysis:
The test samples were analysed directly without further treatment.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:

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 single nominal concentration of 100 mg/l. A single test concentration was used as results from the Acute Toxicity to Daphnia magna study (Safepharm Laboratories Project Number 0345/0911) indicated that toxicity was not expected at this concentration.

An amount (2000 mg) of test material was dissolved directly in dechlorinated tap water and the volume adjusted to 1 litre to give a 2000 mg/l stock solution. The entire volume of the 2000 mg/l stock solution was added to a final volume of 20 litres of dechlorinated tap water and stirred using a flat bladed mixer for approximately 1 minute to give the 100 mg/l test concentration.

The stock solution was inverted several times to ensure adequate mixing and homogeneity.

In the range-finding test 3 fish were added to each 20 litre test and control vessel and maintained at approximately 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 under static test conditions.

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

Each vessel 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.

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/EEC Test Guidelines, no mortalities or sub-lethal effects of exposure were observed.

Experimental Preparation:
For the purpose of the definitive test the test material was dissolved directly in dechlorinated tap water.

An amount (8000 mg) of test material was dissolved in dechlorinated tap water and the volume adjusted to 2 litres to give a 4000 mg/l stock solution. An aliquot (625 ml) of this stock solution was added to a final volume of 25 litres of dechlorinated tap water and stirred using a flat bladed mixer for approximately 1 minute to give the 100 mg/l test concentration. This was conducted in duplicate to give replicates R1 and R2.

The stock solution was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

other: Rainbow trout (Oncorhynchus mykiss)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in house since. Fish were maintained in a glass fibre tank with a "single pass" water renewal system.
- Age at study initiation (mean and range, SD): Juvenile
- Length at study initiation (length definition, mean, range and SD): The fish had a mean standard length of 5.2 cm (sd = 0.2) at the end of the definitive test.
- Weight at study initiation (mean and range, SD): The fish had a mean weight of 1.95 g (sd = 0.26) at the end of the definitive test.
- Food type: The stock fish were fed commercial trout pellets which was discontinued 24 hours prior to the start of the definitive test.
Based on the mean weight value this gave a loading rate of 0.78 g bodyweight/litre.

ACCLIMATION
- Acclimation period: Fish were acclimatised to test conditions from 18 April 2007 to 30 April 2007.
- Acclimation conditions (same as test or not): Same as test conditions.
- Type and amount of food: Commercial trout pellets.
- Health during acclimation (any mortality observed): There was zero mortality in the 7 days prior to the start of the test.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

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.

Test conditions

Hardness:

Test water had a total hardness of approximately 140 mg/l as CaCO3.

Test temperature:

The water temperature was recorded daily throughout the test.
The water temperature was controlled at approximately 14°C.

pH:

pH was recorded daily throughout the test.
pH range: 7.7 - 8.2

Dissolved oxygen:

Dissolved oxygen concentrations were recorded daily throughout the test.
The test water had a dissolved oxygen content of greater than or equal to 8.2 mg O2/l.

Salinity:

Not measured.

Nominal and measured concentrations:

Nominal concentration of 100 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel: 25 litre glass exposure vessels.
- Type: The test vessels were covered to reduce evaporation.
- Material, size, headspace, fill volume: A test media volume of 25 litres was used to ensure that the loading rate used during the definitive test was less than 1 g bodyweight per litre of test medium.
- Aeration: The test vessels were aerated via narrow bore glass tubes.
- Renewal rate of test solution (frequency/flow rate): A semi-static test regime was employed in the test involving a daily renewal of the test preparations to ensure that the concentrations of the test material remained near nominal and to prevent the build up of nitrogenous waste products.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 1 (The control group was maintained under identical conditions but not exposed to the test material).



TEST MEDIUM / WATER PARAMETERS
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.


OTHER TEST CONDITIONS
- Photoperiod: Photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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.


TEST CONCENTRATIONS
- Range finding study
- Test concentrations:
In the range-finding test fish were exposed to a single nominal concentration of 100 mg/l.
- Results used to determine the conditions for the definitive study:
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/EEC Test Guidelines, no mortalities or sub-lethal effects of exposure were observed.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 2

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:
Cumulative mortality data from the exposure of rainbow trout to the test material during the range-finding test are given in Table 1. There were no sub-lethal effects of exposure during the range-finding test.
The results showed no mortalities at the single test concentration of 100 mg/l.
Based on this information, a single test concentration, in duplicate, 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/EEC Test Guidelines, no mortalities or sub lethal effects of exposure were observed.

Definitive Test:
Mortality data:
Cumulative mortality data from the exposure of rainbow trout to the test material during the definitive test are given in Table 2.

There were no mortalities in 20 fish exposed to a test concentration of 100 mg/l for a period of 96 hours.

The results of the definitive test showed the highest test concentration resulting in 0% mortality to be greater than or equal to 100 mg/l, the lowest test concentration resulting in 100% mortality to be greater than 100 mg/l and the No Observed Effect Concentration (NOEC) to be 100 mg/l. The No Observed Effect Concentration is based upon zero mortalities and the absence of any sub-lethal effects of exposure at this concentration.

Sub-lethal effects:
There were no sub-lethal effects of exposure observed in 20 fish exposed to a test concentration of 100 mg/l for a period of 96 hours.

Observations on test material solubility:
Throughout the duration of the test the test concentrations were observed to be clear, yellow solutions.

Physico-chemical measurements:
Temperature was maintained at approximately 14°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

The oxygen concentration in the some of test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed.

Verfication of test concentrations:
Analysis of the test preparations at 0, 24 and 96 hours showed measured test concentrations to range from 99% to 106% of nominal and so it was considered justifiable to estimate the LC50 values in terms of the nominal test concentrations only.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Table 1               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
100	0	0	0	0	0	0

Table 2               Cumulative Mortality Data in the Definitive Test

Nominal Concentration (mg/l)	Cumulative Mortality (Initial Population =10)						% Mortality
	3 Hours	6 Hours	24 Hours	48 Hours	72 Hours	96 Hours	96 Hours
Control	0	0	0	0	0	0	0
100 R1	0	0	0	0	0	0	0
100 R2	0	0	0	0	0	0	0

Physico-Chemical Measurements

Nominal Concentration (mg/l)	Time (Hours)
	0 Hours (Fresh Media)				24 Hours (Old Media)				24 Hours (Fresh Media)
	pH	mg O2/l	%ASV*	TºC	pH	mg O2/l	%ASV*	T°C	pH	mg O2/l	%ASV*	T°C
Control	8.0	10.1	98	14	8.0	9.2	89	14	7.9	10.1	96	13
100 R1	8.0	10.1	98	14	7.9	8.5	83	14	7.9	10.1	98	14
100 R2	8.0	10.2	99	14	7.8	8.5	83	14	7.9	10.0	97	14

 

Nominal Concentration (mg/l)	Time (Hours)
	48 Hours (Old Media)				48 Hours (Fresh Media)				72 Hours (Old Media)
	pH	mg O2/l	%ASV*	TºC	pH	mg O2/l	%ASV*	T°C	pH	mg O2/l	%ASV*	T°C
Control	8.1	9.6	93	14	7.8	10.2	97	13	8.1	10.1	98	14
100 R1	8.1	9.6	93	14	7.8	10.1	96	13	8.1	10.0	97	14
100 R2	7.9	9.5	92	14	7.8	10.3	98	13	8.0	10.0	97	14

 

Nominal Concentration (mg/l)	Time (Hours)
	72 Hours (Fresh Media)				96 Hours (Old Media)
	pH	mg O2/l	%ASV*	TºC	pH	mg O2/l	%ASV*	T°C
Control	7.9	10.4	101	14	8.2	9.6	93	14
100 R1	7.8	10.6	103	14	8.1	9.5	92	14
100 R2	7.7	10.5	102	14	8.1	9.6	93	14

*ASV= Dissolved oxygen concentration expressed as a percentage of Air Saturation Value

R₁and R₂= Replicates 1 and 2

Verification of Test Concentrations

Results

Sample	Nominal Concentration (mg/l)	Concentration Found (mg/l)	Expressed as a Percent of the Nominal Concentration (%)
0 Hours	Control	<LOQ	-
	100 R1	99.0	99
	100 R2	106	106
24 Hours	Control	<LOQ	-
	100 R1	103	103
	100 R2	105	105
96 Hours	Control	<LOQ	-
	100 R1	98.5	99
	100 R2	99.5	99

LOQ = Limit of quantitation

R₁– R₂= Replicates 1 to 2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

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

Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the test material to rainbow trout (Oncorhynchus mykiss). The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods.

Following a preliminary range-finding test fish were exposed, in two groups of ten, to an aqueous solution of the test material, at a single concentration of 100 mg/l for a period of 96 hours at a temperature of approximately 14ºC under semi-static test 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.

The 96-Hour LC₅₀based on nominal test concentrations was greater than 100 mg/l and correspondingly the No Observed Effect Concentration was 100 mg/l.

Analysis of the test preparations at 0, 24 and 96 hours showed measured test concentrations to range from 99% to 106% of nominal and so the results are based on nominal test concentrations only.

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