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Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02/10/2008-10/02/2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to the OECD Guideline 203 and it is GLP

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

ANALYSIS OF THE TEST ITEM CONCENTRATIONS
For the determination of the actual test item concentrations in the test media, duplicate samples were taken from each treatment before the start of the test, after 48 hours and at the end of the test after 96 hours. However, from the three highest nominal test concentrations of 2.2, 4.6 and 10 mg/L the last samples were taken after 48 hours at 2.2 mg/L and after 24 hours at 4.6 and 10 mg/L, since at that time all fish were dead at these concentrations. All samples were taken from the approximate center of the aquaria without mixing the test media.
For the determination of the actual test item concentrations in the application solutions, duplicate samples were taken from each treatment at the start of the test (Day 0) and at the end of the respective exposure periods (Day 1, 2, and 4).
All samples were stored deep-frozen (at about -20 °C) immediately after sampling. Based on preexperiments for investigation of the storage stability (non-GLP), the test item was found to be sufficiently stable in the test water under these storage conditions.

WATER QUALITY CRITERIA
The water temperature, pH and dissolved oxygen concentrations were measured at the start of the test and once daily during the test for each treatment with surviving fish. At the same dates the appearance of the test media was recorded. The total hardness of the water used in the test was measured at the start of the test in the control.

Vehicle:

no

Details on test solutions:

DOSAGE
In this flow-through test, the concentrations of the test item in the test media were maintained by dosing concentrated aqueous test item solutions (= application solutions) into test water by means of automatic dispenser units. The stability of the application solutions during the test periods was determined in a pre-experiment (without GLP) and was verified within this study.
The application solution used for the dosage of the highest test concentration was prepared by completely dissolving 33.34 g of test item in 10 liters of purified water using ultrasonic treatment for 15 minutes and stirring for 15 minutes at room temperature in the dark. Adequate volumes of this intensively stirred application solution were added to purified water and were intensively mixed to prepare the test application solutions used for the dosage of media with lower test
concentrations.
All application solutions were protected from light and were continuously mixed by magnetic stirrers during the test period. They were dosed into mixing vessels (volume about 2 L) using HAMILTON Digital dispenser (HAMILTON, Germany). The dosing intervals were controlled by a timer. The dosing adjustments were: 36 mL of application solution per hour (20 dosages per hour, 1.8 mL per dosage). Test water continuously flowed into the mixing vessels at a rate of 288 liters per 24 hours (corresponding to 12 liters per hour), regulated by flow-meters. In the mixing vessels, the prepared test media were continuously stirred and flowed out into the aquaria. The test media volumes in the aquaria amounted to about 48 liter, thus, the flow-rate through the aquaria was equivalent to a six-fold theoretical volume exchange per day. This flow-rate ensured a frequency of water removal at any one time during the test of at least 6 liters/g fish/day, as
recommended by the test guideline.
Just before introduction of the fish (= start of the test), the test item was mixed directly into the aquaria (144 mL of each application solution per 48 liters of test water). Then, the above described dosing system was started.

Test Water
Local tap water (non chlorinated well water of drinking water quality) was used, reduced for total hardness by ion exchange

Material
One glass test vessel with 48 liters of test medium was used for each treatment.
The test vessels were labeled with the study number and all necessary additional information to
ensure unique identification.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout
- Source: P.Hohler, 4314 Zeiningen/Switzerland
- Length at study initiation (length definition, mean, range and SD): 5.5±0.19
- Weight at study initiation (mean and range, SD): 1.7±0.23g

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

214 mg/L CaCO3

Test temperature:

14ºC

pH:

8.4

Dissolved oxygen:

9.7mg/L

Nominal and measured concentrations:

Nominal concnetration: 0.46, 1.0, 2.2, 4.6 and 10 mg/L
Additionally, a control (test water without test item) was tested in parallel. A flow-through test design was chosen. The selection of the test concentrations was based on the resultsof a range-finding test (non-GLP).

Details on test conditions:

Experimental Conditions
The water temperature in the test vessels was maintained at 14 °C (Table 5). The test vessels were slightly aerated during the test period.
A 16-hour light to 8-hour dark photoperiod, with a 30-minute transition period was used (light intensity during the light period was approximately within the range of 70 to 500 Lux 100 to 560 Lux). The test duration was 96 hours and the fish were not fed during the test.

At the start of the test, 7 fish were introduced into each test vessel in a random order. The loading rate was 0.25 g fish wet weight per liter test medium. Thus, the requirements of a loading rate of less than 1 g fish/L was fulfilled.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.93 mg/L

Conc. based on:

test mat.

Remarks on result:

other: 0.62-1.4 mg/L

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

0.43 mg/L

Conc. based on:

test mat.

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

1.8 mg/L

Conc. based on:

test mat.

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.43 mg/L

Conc. based on:

test mat.

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

0.87 mg/L

Conc. based on:

test mat.

Sublethal observations / clinical signs:

Nominal test item concentration (mg/L)	Mean measured test item concnetration (mg/L)
0,46	0,43	93% of nominal
1	0,87	87% of nominal
2,2	1,8	81% of nominal
4,6	3,9	85% of nominal
10	7,6	76% of nominal

Validity criteria fulfilled:

yes

Conclusions:

The biological test results after 96 hours test duration, based on mean measured concentrations was LC50(96h)=0.93 mg/L

Executive summary:

The acute toxicity of the registered substance to rainbow trout (Oncorhynchus mykiss) was determined in a 96-hour flow through test according to Commission Regulation (EC) No 440/2008, Part C.1 and the OECD Guideline for Testing of Chemicals No. 203, (1992). The test was compliant with GLP.

The nominal test item concentrations tested were 0.46, 1.0, 2.2, 4.6 and 10 mg/L. Additionally a control group was tested in parallel. The analytically determined concentrations of the test item in the test media varied in the range from 72 to 93% of the nominal values during the test period. The mean measured test item concentrations (calculated as the arithmetic mean over all measurements per test concentration) ranged from 76 to 93% of the nominal values. All reported biological results are related to the mean measured concentrations of the test item.

The biological test results after 96 hours test duration, based on mean measured concentrations are shown hereafter:

– 96-hour LC50: 0.93 mg/L (95% confidence interval: 0.62 – 1.4 mg/L)

– 96-hour LC0: 0.43 mg/L

– 96-hour LC100: 1.8 mg/L

– 96-hour NOEC: 0.43 mg/L

– 96-hour LOEC: 0.87 mg/L

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

06/09/1991-11/10/1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to the OECD Guideline 203 but it is not GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

no

Analytical monitoring:

not specified

Vehicle:

no

Test organisms (species):

Leuciscus idus melanotus

Details on test organisms:

Length: 5 - 8 cm

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

20ºC

pH:

8.31

Dissolved oxygen:

7.80 mg/L

Nominal and measured concentrations:

2; 3 and 4 mg/L

Details on test conditions:

Test conditions: Static test for 96 hours at 20±1ºC, 10 fishes per test (1 fish per litre).
Tests are performed in drinking water.

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

1 mg/L

Conc. based on:

test mat.

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

3 mg/L

Conc. based on:

test mat.

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

4 mg/L

Conc. based on:

test mat.

Details on results:

The LC50 value was determined by probit analysis

Sublethal observations / clinical signs:

Range Finding Test Beginning Completion:

Concentration of the test substance mg/L	Effect on fish mortality after 48h
1	0
10	100
100	100

Definitive Test Beginning Completion

Concentration of the test substance mg/L	Effect on fish mortality after
	24h	48h	72h	96h
2	0	0	0	20
3	0	0	0	20
4	100	-	-	-

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the LC50 (96h) of the test item to fish (Leuciscus idus melanotus) was found to be 3 mg/L.

Executive summary:

The acute toxicity of the test item to fish ( Leuciscus idus melanotus) was determined in a non-GLP 96-hour test according to the OECD Guideline for Testing of Chemicals No. 203.

The test conditions were as follows: static test for 96 hours at 20±1ºC, 10 Fishes per test (1 fish per litre). The fishes were exposed to various concentrations of the test substance. Mortality was recorded after 24, 48, 72 and 96 hours. Dead fish were instantly removed from the test tank. The preparation of stock solution with a concentration of 1g/L of test susbtance was done by dilution with drinking water.

Under the test conditions, the LC50 (96h) of the test item to fish (Leuciscus idus melanotus) was found to be 3 mg/L.

In addition, the LC0 and the LC100 were 1 and 4 mg/L respectively.

Description of key information

A study was available to assess the acute toxicity of the test item to rainbow trout in a 96 -hours flow through test according to the OECD Guideline 203 and in compliance with GLP.

The biological test results based on mean measured concentrations was LC50(96h)=0.93 mg/L

This study was considered acceptable for that endpoint.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.93 mg/L

Additional information

Moreover, a supporting study that assesses the acute toxicity of the test item to the fish Leuciscus idus melanotus has been added to the dossier. This non-GLP study was performed according to the OECD Guideline for Testing of Chemicals No. 203.

Under the conditions of this test, the LC50 (96h) of the test item to fish (Leuciscus idus melanotus) was found to be 3 mg/L.