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Long-term toxicity to fish

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Reference
Endpoint:
fish, juvenile growth test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02/10/2008-09/03/2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD 215 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 215 (Fish, Juvenile Growth Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.14 (Fish Juvenile Growth Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
ANALYSIS OF THE TEST ITEM CONCENTRATIONS
For the determination of the test item concentrations, duplicate samples from all test concentrations and from the control were taken at the start of the test (0 hours) and on Days 4, 8, 11, 14, 18, 23, 25 and 28 of the exposure period. The last samples from the highest nominal test concentration of 1.0 mg/L were taken on Day 8, since all fish were dead at that time at this test concentration.
All test medium samples were taken from the approximate centre of the aquaria without mixing of the test media.
For the analysis of the test item concentrations in the application solutions and for determination of their stability, duplicate samples were taken at the start and end of two application solution renewal periods (renewal periods of 7 and 8 days).
All samples were deep frozen (at about -20 °C) immediately after sampling. Based on preexperiments for investigation of the storage stability (without GLP) the test item is stable in the test water under the storage conditions.
The actual concentration of the test substance was measured in all duplicate test medium samples from the nominal concentrations of 0.32 and 1.0 mg/L, which were determined as the NOEC and LOEC in this test. From the corresponding application solutions one of the duplicate samples was analyzed.
The concentrations below the NOEC were not analyzed, because they were not relevant for the interpretation of the biological results.
From the control samples, one of the duplicate samples of the sampling dates on Days 0, 8 and 28 was analyzed.

WATER QUALITY CRITERIA
The water temperature, pH values and the dissolved oxygen concentrations in the test media of all test concentrations and the control were measured at least two times per week. The water hardness of the test water was measured once per week during the test. The appearance of the test media was recorded daily.
Vehicle:
no
Details on test solutions:
The following nominal test item concentrations were tested: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. Additionally, a control (test water without test item) was tested in parallel.
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 application solutions were prepared immediately before the test start and were renewed every 7 to 8 days (on Day 0, 8, 15 and 22). The stability of the test item in the application solutions during the renewal periods was determined in a pre-experiment (without GLP) and was verified within this study.
At the start of the test and at Day 8, the application solution used for the dosage of the highest test concentration was prepared by completely dissolving 3375 mg of the test item (range: 3376.0 and 3378.2 mg) in 4500 mL of purified water. At Day 15 and 22, when all fish were dead in the highest test concentration, the application solution used for the dosage of the second highest test concentration was prepared by dissolving 1500 mg test item (range: 1500.2 and 1500.8 mg) in 2000 mL of purified water. For this, ultrasonification for 15 minutes and intense stirring for 15 minutes were applied. These application solutions were diluted with purified water to prepare the application solutions used for the dosage of the 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: 16 mL of application solution per hour (20 dosages per hour, 0.80 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 (64 mL of each application solution per 48 liters of test water). Then, the dosing system as described above was started.
The dosing system was calibrated before test start and was controlled at least once per day. The actual concentrations of the test item in the test media determined in the test as LOEC and NOEC and in the corresponding application solutions were analytically determined.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Strain: Oncorhynchus mykiss
- Source: P.Hohler, Zeiningen
- Age at study initiation (mean and range, SD):
- Weight at study initiation (mean and range, SD): 1.2 g
- Acclimatization: 7 days

FEEDING DURING TEST
- Food type: commercial fish diet (HOKOVIT 502)
- Amount: 2% during the acclimatization period and 4% during the first week of the definitive test.
- Light: 16-hour light to 8-hour dark
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
28 d
Hardness:
223-232 mg/L as CaCO3
Test temperature:
13.2-13.9 ºC
pH:
7.6-8.0
Dissolved oxygen:
9.9 mg/L
Nominal and measured concentrations:
Nominal: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L.
The mean measured test item concentrations (calculated as the arithmetic mean over all measurements per test concentration) were 0.15 mg/L (nominal 0.32 mg/L) and 0.45 mg/L (nominal 1.0 mg/L)
Details on test conditions:
One 60-litre aquarium with about 48 liters of test medium was used for each test concentration and the control. At the start of the test (Day 0), 10 fish were introduced into each aquarium in a random order. The test vessels were labeled with the Study number and all necessary additional information to ensure unmistakable identification. Uneaten food and fish feces were removed regularly. The test media were slightly aerated during the test. The loading rate of the test fish did not exceed 1 g fish wet weight per liter medium at the end of the test.

- Test duration: 28 days
- Water temperature: 13.2-13.9 °C (with a max. difference of ± 0.4 °C between the aquaria at any one time during the test).
- Light: A 16-hour light to 8-hour dark photoperiod, light intensity of approximately 240–520 Lux (with a 30-minute transition period).
- Test water: Local tap water (non chlorinated well water of drinking water quality), reduced for total hardness by ion exchange was used. The total hardness was between 223 and 232 mg/L as CaCO3. The test water was aerated prior to the preparation of the test media until oxygen saturation was reached.

During holding, acclimatization period and definitive test, the fish were daily fed with a commercial fish diet (HOKOVIT 502, 1.2 mm, supplied by H.U. Hofmann AG, CH-4922
Bützberg, Switzerland).
The daily food amount (dry weight related to the mean initial fish wet weight in each treatment) was 2% during the acclimatization period and 4% during the first week of the definitive test.
Every subsequent week, the food amount fed to the fish was increased by 25%. The daily food ration was divided into two equal portions and fed to the fish in two feeding intervals per day (separated by more than five hours) during the light period. Food was withheld from fish for 24 hours prior to weighing at test end.
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
0.15 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
0.45 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Duration:
28 d
Dose descriptor:
other: LLC
Effect conc.:
0.45 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
28 d
Dose descriptor:
other: LLC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Details on results:
Biological results
In the control and at the test item concentrations up to and including 0.15 mg/L (nominal 0.32 mg/L) no significant number of fish died during the test period and no visible abnormalities were observed. A mortality rate of 10% as was not estimated as a toxic effect since it was also observed in the control and according to test guideline a mortality rate of 10% is accepted in the control for the test to be valid.
At the test concentration of nominal 0.10 mg/L, two of the ten test fish had died. This was not attributed to a toxic effect of the test item, as no other visible abnormalities were observed in the fish, and at the next higher test concentration of 0.15 mg/L (nominal 0.32 mg/L) (which was determined as the NOEC in this test), only one fish died during the test. Therefore, no concentration-effect relationship for fish mortality was observed up to the test concentration of 0.15 mg/L (nominal 0.32 mg/L).
At the highest test concentration of 0.45 mg/L (nominal 1.0 mg/L), all fish were dead after 8 days of test duration.
The body wet weight of the test fish measured at test start and at the end of the test after 28 days are listed in Table 2. At the start of the test, the mean wet weight of the test fish, used at each test concentration and the control, were identical. The range of individual fish wet weight was between 1.1 and 1.3 g in all treatments and, thus, within ±10% of the mean value as requested by the guideline. During the test period of 28 days, the mean wet weight of the test fish in the control had increased by a factor of 3.2 (a minimum factor of 1.5 is requested by the test guideline).
At the end of the test period of 28 days, the mean fish wet weight (Table 2) and the pseudo specific growth rate r’ for fish wet weight (Table 3) were nearly identical or even slightly higher compared to the control (range: 91 - 111% of control) up to the test concentration of 0.15 mg/L (nominal 0.32 mg/L) and no concentration effect relationship was observed. Both test parameters were not statistically significantly different to the control at all test concentrations (results of
Williams’ tests, one-sided smaller, α = 0.05).
Therefore, based on mortality, observations for visible abnormalities, fish wet weight, and pseudo specific growth rate r’ for fish wet weight, the highest concentration tested without toxic effects (NOEC) observed at rainbow trout after an exposure period of 28 days was determined to be mean measured 0.15 mg/L (nominal 0.32 mg/L).
The LOEC (lowest test concentration at which a significant toxic effect on the test organisms is observed) was determined to be mean measured 0.45 mg/L (nominal 1.0 mg/L) due to the 100% mortality of the test fish at this test concentration.

Table: Mortality and visible abnormalities

  Nominal test item concentration (mg/L)
Day control 0,01 0,032 0,1 0,32
(0,15)*
1
(0,45)*
  Number of abnormal and deathfish / number of dead fish
  Type of visible abnormalities
3 hour 0/0 0/0 0/0 0/0 0/0 0/0
1 0/0 0/0 0/0 0/0 0/0 10/1
AP, BO, TS
2 0/0 0/0 0/0 0/0 0/0 10/2
AP, BO, SR, TS
3 0/0 0/0 0/0 0/0 0/0 10/3
AP, BO, SR, TS
4 0/0 0/0 0/0 0/0 0/0 10/5
AP, BO, SR, TS
5 0/0 0/0 0/0 0/0 0/0 10/6
'AP/SR
6 0/0 0/0 0/0 0/0 0/0 10/8
AP, SR
7 0/0 0/0 0/0 0/0 0/0 10/9
AP/SR
8 0/0 0/0 0/0 0/0 0/0 10/10
9 0/0 0/0 0/0 0/0 0/0 -
10 0/0 0/0 0/0 1/1 0/0 -
11 0/0 0/0 0/0 1/1 0/0 -
12 0/0 0/0 0/0 1/1 0/0 -
13 0/0 0/0 0/0 2/2 0/0 -
14 1/1 0/0 0/0 2/2 1/1 -
15 1/1 0/0 0/0 2/2 1/1 -
16 1/1 0/0 0/0 2/2 1/1 -
17 1/1 0/0 0/0 2/2 1/1 -
18 1/1 0/0 0/0 2/2 1/1 -
19 1/1 0/0 0/0 2/2 1/1 -
20 1/1 0/0 0/0 2/2 1/1 -
21 1/1 0/0 0/0 2/2 1/1 -
22 1/1 0/0 0/0 2/2 1/1 -
23 1/1 0/0 0/0 2/2 1/1 -
24 1/1 0/0 0/0 2/2 1/1 -
25 1/1 0/0 0/0 2/2 1/1 -
26 1/1 0/0 0/0 2/2 1/1 -
27 1/1 0/0 0/0 2/2 1/1 -
28 1/1 0/0 0/0 2/2 1/1 -

* mean measured concentration

- all fish were dead

Abbreviations: AK: strongly extended gills KR: convulsions AP: apathy OB: fish mainly at the water surface BA: distended abdomen SA: mucus secretion BO: fish mainly at the bottom SR: fish lying on side or back on the bottom of the test vessel SV: strong ventilation GA: exophthalmus TS: tumbling during swimming KF: cessation of food intake VF: changed body color

Analytical Results

The analytical results are presented in Table 2 of the attached analytical part report. The concentrations of test substance in the freshly prepared and aged application solutions ranged from 96 to 107% of the nominal value. This shows the correct preparation of the application solutions and the stability of the test item in the application solutions during the renewal periods of 7 and 8 days.

The test substance concentrations in the aged analyzed test media of nominal 1.0 and 0.32 mg/L varied in the range of 36 and 53% of nominal during the test period. The mean measured test item concentrations (calculated as the arithmetic mean over all measurements per test concentration) were 0.15 mg/L (nominal 0.32 mg/L) and 0.45 mg/L (nominal 1.0 mg/L). The reported biological results (the NOEC and LOEC) are based on the mean measured concentrations of the test item.

Water Quality Criteria

No remarkable observations were made concerning the appearance of the test media.

All test media were clear solutions throughout the whole test period.

During the test period, the pH values in the test media were between 7.6 and 8.0. The dissolved oxygen concentrations were always 9.4 mg/L or higher, and thus, higher than 60% oxygen saturation.

The water temperatures measured in all treatments were between 13.2 and 13.9 °C. Consequently, the difference in water temperature was

lower than ± 1.0°C between the aquaria at any time during the test period, as requested by the guideline. The mean water temperatures measured in all treatments were between 13.5 and 13.6 °C.

Validity criteria fulfilled:
yes
Conclusions:
Based on mortality, visible abnormalities, fish wet weight, and pseudo specific growth rate for fish wet weight, the highest concentration tested without toxic effects (NOEC) observed at rainbow trout after an exposure period of 28 days was determined to be 0.15 mg/L. The lowest concentration tested with toxic effects (LOEC) and the LLC amounted to 0.45 mg/L due to the 100% mortality of the test fish at this concentration. The reported biological results are based on the mean measured concentrations of the test item.
Executive summary:

The toxicity of the test item of the substance to rainbow trout (Oncorhynchus mykiss) was investigated in a 28 -day flow-through test according to the EU Commission Directive 2001/59/EC, Part C.14 and the OECD Guideline for Testing of Chemical, No 215, "Fish, Juvenile Growth Test". The study was performed under GLP compliance.

The nominal test item concentration tested were 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. Additionally, a control group was tested in parallel. The concentrations of the substance in the freshly prepared and aged application solutions ranged from 96 to 107% of the nominal value. This shows the correct preparation of the application solutions and the stability of the test item in the application solutions during the renewal periods of 7 and 8 days. The substance concentrations in the aged analyzed test media of nominal 1.0 and 0.32 mg/L varied in the range of 36 and 53% of nominal during the test period. The mean measured test item concentrations (calculated as the arithmetric mean over all measurements per test concentration) were 0.15 mg/L (nominal 0.32 mg/L) and 0.45 mg/L (nominal 1.0 mg/L). The biological result are based on the mean measured concentrations of the test item.

Based on mortality, visible abnormalities, fish wet weight, and pseudo specific growth rate for fish wet weight, the higuest concentration tested toxic effects (NOEC) observed at rainbow trout after an exposure period of 28 days was determined to be mean measured 0.15 mg/L. The lowest concentration tested with toxic effects (LOEC) and the LLC amounted to 0.45 mg/L due to the 100% mortality of the test fish at this concentration.

Description of key information

In order to assess the long-term toxicity of the registered substance to fish, a reliable study was available. This study has investigated the toxicity of the test item to rainbow trout (Oncorhynchus mykiss), in a 28 -day flow-through test according to the EU Commission Directive 2001/59/EC, Part C.14 and the OECD Guideline for Testing of Chemical, No 215, "Fish, Juvenile Growth Test". The study was done under GLP conditions.

Based on mortality, visible abnormalities, fish wet weight, and pseudo specific growth rate for fish wet weight, the highest concentration tested toxic effects (NOEC) observed at rainbow trout after an exposure period of 28 days was determined to be 0.15 mg/L. The lowest concentration tested with toxic effects (LOEC) and the LLC amounted to 0.45 mg/L due to the 100% mortality of the test fish at this concentration. The reported biological results are based on the mean measured concentrations of the test item.

The study was considered acceptable for that endpoint.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
0.15 mg/L

Additional information

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