HPSA

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:

22 - 26 April 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well documented 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)

Remarks:

The Department of Health of the Government of the United Kingdom

Analytical monitoring:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Range-finding study :
The test concentrations to be used in the definitive study were determined by a preliminary range-finding study.
In the range-finding study fish were exposed to a series of nominal test concentrations of 1.0, 10, 100 and 1000 mg/I. The test material was dispersed directly in water. An amount of test material (4.0 g) was dissolved in dechlorinated tap water and the volume adjusted to 1 litre to give a 4.0 g/I aqueous stock solution. Aliquots (5.0, 50 and 500 ml) of this stock solution were each separately dispersed into dechlorinated tap water and the volume adjusted to 20 litres to give the 1.0, 10 and 100 mg/I test concentrations. To prepare the 1000 mg/I test concentration an amount of test material (10 g) was dispersed directly in 10 litres of dechlorinated tap water. A 10 litre test volume was used at this concentration to minimise the amount of test material used.
Definitive study :
Based on the results of the range-finding study the following test concentrations were assigned to the definitive study: 100;180, 320, 560 and 1000 mg/I. For the purpose of the definitive study the test material was prepared by direct dispersion in water. Amounts of test material (2.0, 3.6, 6.4, 11.2 and 20 g) were each separately dispersed directly into 20 litres of dechlorinated tap water to give the test series of 100, 180, 320, 560 and 1000 mg/I respectively.

Test organisms (species):

Leuciscus idus

Details on test organisms:

TEST ORGANISM
- Common name: Golden orfe
- Source: Xotic Fish Centre, Alfreton, Derbyshire, UK
- Age at study initiation (mean and range, SD): juvenile fish
- Length at the end of the definitive study : mean standard length of 5.4 cm (sd = 0.3)
- Weight at the end of the definitive study : mean weight of 1.54 g (sd = 0.38)
- Feeding during test: none
- Food type: commercial Tetramin® flake food (discontinued 48 h prior to the start of the definitive test)

ACCLIMATION
- Acclimation period: 15 - 22 April 1996
- Acclimation conditions: 16 h light and 8 h darkness cycle
- Health during acclimation (any mortality observed): no mortality in the 7 days prior to the start of the test

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

100 mg/L as CaCO3

Test temperature:

21 +/- 1.0 °C

pH:

100 mg/l : 7.6 - 7.9
180 mg/l : 7.3 - 7.9
320 mg/l : 6.2 - 7.3
560 mg/l : 3.6
1000 mg/l : 2.6

Dissolved oxygen:

6.8 - 8.8 mgO2/l

Nominal and measured concentrations:

Nominal concentrations : 100, 180, 320, 560 and 1000 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: glass vessels with a "single pass" water renewal system
- Type: covered (to reduce evaporation)
- Material, size, headspace, fill volume: 20 L
- Aeration: via narrow bore glass tubes
- Renewal rate of test solution: daily
- No. of organisms per vessel: 10 fish
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- No. of vessels per vehicle control: 1
- Biomass loading rate: 0.77 g bodyweight/L
-Any mortalities and adverse reactions to 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 WATER
Laboratory test water was dechlorinated by passage through an activated carbon filter giving water with a total hardness of approximately 100 mg/L as CaCO3.

OTHER TEST CONDITIONS
- Adjustment of pH: not adjusted, just measured
- Photoperiod: 16 h light and 8 h darkness

Reference substance (positive control):

not required

Remarks:

according to OECD 203

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

420 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL : 320-560 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

180 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

The results of the definitive study showed the highest test concentration resulting in 0% mortality to be 320 mg/L, the lowest test concentration resulting in 100% mortality to be 560 mg/L and the No Observed Effect Concentration (NOEC) to be 180 mg/L. The No Observed Effect Concentration is based upon zero mortalities and the absence of any sub-lethal responses to exposure at this concentration. The only sub-lethal response to exposure (other than death) was observed at test concentrations of greater than 180 mg/L. This response was fish swimming at the surface. The LC50 value of 420 mg/L (420 ppm = 4.2 x 10E-4) corresponds to an evaluation number (Bewertungszahl, BWZ) for the German Water Hazard Classification Scheme of 3.4 .

Results with reference substance (positive control):

Not applicable.

Sublethal observations / clinical signs:

Range-finding Study :

Cumulative mortality data from the exposure of golden orfe to the test material during the range-finding study are given in Table 1. There were no adverse reactions to exposure (other than death) during the range-finding study. The results showed no mortalities at the test concentrations of 1.0, 10 and 100 mg/L. However, mortalities were observed at 1000 mg/L.

TABLE 1

CUMULATIVE MORTALITY DATA IN THE RANGE-FINDING STUDY

Nominal concentration (mg/L)	Cumulative mortality (Initial population = 3)
	24 hours	48 hours	72 hours	96 hours
Control
1,0	0	0	0	0
10	0	0	0	0
100	0	0	0	0
1000	3	3	3	3

Definitive Study :

Cumulative mortality data from the exposure of golden orfe to the test material during the definitive study are given in Table 2. The results of the definitive study showed the highest test concentration resulting in 0% mortality to be 320 mg/L, the lowest test concentration resulting in 100% mortality to be 560 mg/L and the No Observed Effect Concentration (NOEC) to be 180 mg/L. The No Observed Effect Concentration is based upon zero mortalities and the absence of any sub-lethal responses to exposure at this concentration.

TABLE 2

CUMULATIVE MORTALITY DATA IN THE DEFINITIVE STUDY

Nominal concentration (mg/L)	Cumulative mortality (Initial population = 3)						% 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
180	0	0	0	0	0	0	0
320	0	0	0	0	0	0	0
560	6	10	10	10	10	10	100
1000	10	10	10	10	10	10	100

TABLE 3

SUB-LETHAL EFFECTS OF EXPOSURE IN THE DEFINITIVE STUDY

Nominal concentration (mg(L)	Sub-lethal Effects *	Time (hours)
		3	6	24	48	72	96
Control	No abnormalities detected
100	No abnormalities detected
180	No abnormalities detected
320	Swimming at surface		4/10	4/10	6/10	6/10	6/10
560	Swimming at surface	4/4	A/D
1000		A/D

* Unless stated no adverse reactions were observed

A/D = All fish dead

Analysis of the mortality data by the moving average method of Thompson (1947) based on the nominal test concentrations gave the following results:

Time (h)	LC50 (mg/l)	95% Confidence Limits (mg/l)
3	530	440 - 640
6	420	320 - 560*
24	420	320 - 560*
48	420	320 - 560*
72	420	320 - 560*
96	420	320 - 560*

*Concentrations resulting in 0 and 100% mortality respectively

Validity criteria fulfilled:

yes

Remarks:

The experiment was conducted in accordance with OECD Guideline 203.

Conclusions:

The study is regarded as a valid guideline study with certificated GLP compliance. The 96-Hour LC50 based on nominal test concentrations was 420 mg/I with 95% confidence limits of 320 - 560 mg/I. The No Observed Effect Concentration was 180 mg/l. The LC50 value of 420 mg/I (420 ppm = 4.2 x 10-4) corresponds to an evaluation number (Bewertungszahl BWZ) for the German Water Hazard Classification Scheme of 3.4.

Executive summary:

The acute toxicity to fish of the test item was tested according to OECD guideline 203 and EU Method C.1 in a semi-static freshwater test with golden orfe (Leuciscus idus) as test organisms, which is a freshwater fish representative of a wide variety of natural habitats, and can therefore be considered as an important non-target organism in freshwater ecosystems (Wetton, 1996). The study was conducted under certificated GLP compliance. The test concentration to be used in the definitive test was determined by a preliminary range-finding test. Based on these range-finding study, the definitive test was conducted at concentrations of 100, 180, 320, 560 and 1000 mg/L. For both purposes the test material was prepared by direct dispersion in dechlorinated tap water. The acclimatisation period of the juvenile fish was conducted for 7 days. The stock fish were fed commercial Tetramin® flake food which was discontinued 48 h prior to the start of the definitive test. There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 5.4 cm (sd = 0.3) and a mean weight of 1.54 g (sd = 0.38) at the end of the definitive test. The test vessels were covered to reduce evaporation and maintained at 21 +/- 1 °C with a photoperiod of 16 h light and 8 h darkness for a period of 96 h. The test vessels were aerated via narrow bore glass tubes. A semi-static test regime was employed 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. The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test. The results of the definitive study showed the highest test concentration resulting in 0% mortality to be 320 mg/L, the lowest test concentration resulting in 100% mortality to be 560 mg/L and the No Observed Effect Concentration (NOEC) to be 180 mg/l. The No Observed Effect Concentration is based upon zero mortalities and the absence of any sub-lethal responses to exposure at this concentration.The only sub-lethal response to exposure (other than death) was observed at test concentrations of greater than 180 mg/L. This response was fish swimming at the surface.

The 96-Hour LC50 based on nominal test concentrations was 420 mg/L with 95% confidence limits of 320 - 560 mg/L. The No Observed Effect Concentration was 180 mg/L.

The LC50 value of 420 mg/L (420 ppm = 4.2 x 10-4) corresponds to an evaluation number (Bewertungszahl BWZ) for the German Water Hazard Classification Scheme of 3.4.

Description of key information

Leuciscus idus (Golden orfe), OECD 203, LC50(96h) : 420 mg/L

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

420 mg/L

Additional information

The acute toxicity to fish of the test item was tested according to OECD guideline 203 and EU Method C.1 in a semi-static freshwater test with golden orfe (Leuciscus idus) as test organisms, which is a freshwater fish representative of a wide variety of natural habitats, and can therefore be considered as an important non-target organism in freshwater ecosystems (Wetton, 1996). The study was conducted under certificated GLP compliance. The test concentration to be used in the definitive test was determined by a preliminary range-finding test. Based on these range-finding study, the definitive test was conducted at concentrations of 100, 180, 320, 560 and 1000 mg/L. For both purposes the test material was prepared by direct dispersion in dechlorinated tap water. The acclimatisation period of the juvenile fish was conducted for 7 days. The stock fish were fed commercial Tetramin® flake food which was discontinued 48 h prior to the start of the definitive test. There was no mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 5.4 cm (sd = 0.3) and a mean weight of 1.54 g (sd = 0.38) at the end of the definitive test. The test vessels were covered to reduce evaporation and maintained at 21 +/- 1 °C with a photoperiod of 16 h light and 8 h darkness for a period of 96 h. The test vessels were aerated via narrow bore glass tubes. A semi-static test regime was employed 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. The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout the test.Theresults of the definitive study showed the highest test concentration resulting in 0% mortality to be 320 mg/L, the lowest test concentration resulting in 100% mortality to be 560 mg/L and the No Observed Effect Concentration (NOEC) to be 180 mg/l. The No Observed Effect Concentration is based upon zero mortalities and the absence of any sub-lethal responses to exposure at this concentration.The only sub-lethal response to exposure (other than death) was observed at test concentrations of greater than 180 mg/L. This response was fish swimming at the surface.

The 96-Hour LC50 based on nominal test concentrations was 420 mg/L with 95% confidence limits of 320 - 560 mg/L. The No Observed Effect Concentration was 180 mg/L.

The LC50 value of 420 mg/L (420 ppm = 4.2 x 10-4) corresponds to an evaluation number (Bewertungszahl BWZ) for the German Water Hazard Classification Scheme of 3.4.