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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study plan start: 26 September 2005, study plan completed: 11 November 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.
Reason / purpose:
reference to other study
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 7346-1 (Determination of the Acute Lethal Toxicity of Substances to a Freshwater Fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] - Part 1: Static Method)
Deviations:
no
Principles of method if other than guideline:
The study also followed the Guidance document on aquatic toxicity testing of difficult test substances and mixtures. OECD series on testing and assessment number 23, December 14, 2000.
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
Final limit test: loading rate of 100 mg/l and blank control.

- Sampling method:
During the final test duplicate samples were taken from the EA-3098 filtrate and the blank control for analysis of test concentrations.
Sampling frequency: At 0 h and 96 h
Sampling volume: 2 ml from the approximate centre of the test vessels.

- Sample storage conditions before analysis:
Samples were stored in a freezer until analysis.

In addition the filter containing the residue was stored for chemical analysis.
Vehicle:
yes
Details on test solutions:
Preparation of test solutions:
The standard test procedures required generation of test solutions that contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturbed the test system was prevented (e.g. film of the test substance on the water surface).

The water solubility of EA3098 was at the highest < 1.0 x 10-4 g/l. Preparation of test solutions started with weighed amounts of test substance that were quantitatively added to volumetric flasks. In the range finding test the solutions were treated with ultra-sonic waves for 10 minutes and then stirred for 1/2 hour before filtering through a 0.45 µm filter (Schleicher and Schuell RC55).
In the final test preparation started with quantitatively adding weighed amounts of test substance to MiIIi-RO water followed by treatment with ultra-sonic waves for 10 minutes (three separate solutions of each 500 mg/l in 5 Iitres). Subsequently, these solutions were stirred for 72 hours and then treated with ultra-sonic waves for 5 minutes before filtering through a 0.45 µm filter (Schleicher and Schuell RC55). Then the filtrates were combined and the salts for the ISO-medium were added to obtain the final test solution.

Range-finding test:
A range-finding test was performed to provide information about the range of concentrations to be used in the final test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
- Three fish per concentration were exposed to filtrates prepared at 0.1, 1, 10 and 100% solution of the filtrate prepared at 100 mg/l.
- Dissolved oxygen concentrations, pH and temperature were only measured in the lowest and highest concentration.
- Samples (2 ml) were taken from all concentrations for chemical analysis at 0h, 24h and 96h.
- No feeding of the fish for 48 prior to the test and during the total test period.

Final test:
Test concentrations: EA-3098 - a filtrate prepared at a nominal loading rate of 100 mg/l.

Control: - Test medium without test substance or other additives (blank-control).






Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
Species: Carp (Cyprinus carpio, Teleostei, Cyprinidae) Linnaeus, 1758
Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, the Netherlands.
Mean length: Range-finding test: 2.6 ± 0.3 cm, final test: 3.1 ± 0.2 cm
Mean weight: Range-finding test: 0.57 ± 0.17 g, final test: 0.87 ± 0.15 g
Characteristics: F1 from a single parent pair bred in UV-treated water.
Total fish used: 26

ACCLIMATION
Acclimatisation: At least 12 days after delivery
Measurements: pH, nitrate and nitrite concentration and ammonia concentration: once a week. Temperature: every day.
In addition, pH and temperature were measured before transferring the fish to the test system.
Feeding: Daily with pelleted fish food and/or artemia
Validity of batch: In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%.









Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
Not applicable.
Test temperature:
Temperature was recorded dally in all vessels, beginning at the start of the test (day 0).
Temperature ranged from 21.5 to 22.0ºC.
pH:
pH was recorded daily in all vessels, beginning at the start of the test (day 0).
pH ranged from 7.4 to 8.0
Dissolved oxygen:
Dissolved oxygen was recorded daily in all vessels, beginning at the start of the test (day 0).
Dissolved oxygen ranged from 7.1 - 8.0 mg O2/l
Nominal and measured concentrations:
Limit test: Nominal loading rate of 100 mg/l.
Measured: Measured concentrations of all three peaks were below their limits of detection.
Details on test conditions:
TEST PROCEDURE AND CONDITIONS:
Test vessels: 20 litres, all-glass, containing 15 litres of test solution.
Number of fish: 7 fish per concentration and control.
Loading: 0.41 g fish/litre i.e. 7 fish per 15 litres of test medium.
Illumination: 16 hours photoperiod daily.
Aeration: The test media were not aerated during the test, except for 1.5 h in the filtrate on day 3.
Feeding: No feeding from 24 hours prior to the test and during the total test period.
Introduction of fish: Within 1/2 hour after preparation of the test media from a holding tank with comparable water quality parameters and pH and temperature differences between test and holding tank media of less than 0.5 unit and 0.5ºC
Euthanasia: At the end of the test the surviving fish were radily killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.

TEST MEDIUM / WATER PARAMETERS
ISO-medium, formulated using Milli-RO water (tap-water purified by reverse osmosis) with the following composition:
CaCl2.2H20: 293.8 mg/l
MgSO4.7H20: 123.3 mg/l
NaHCO3: 64.8 mg/l
KCl: 5.8 mg/l


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mortalities and other effects were measured and recorded at 3, 24, 48, 72 and 96 hours following the start of exposure. In addition, every afternoon from day 0 and every morning from day 1 observation for any dead or severely distressed fish.
Reference substance (positive control):
yes
Remarks:
pentachlorophenol
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.1 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Remarks:
(loading rate)
Basis for effect:
mortality
Remarks on result:
other: The 96h LC50 exceeds the maximum solubility of EA-3098 in water, i.e. at a highest level of 1.0 x 10-4 g/l.
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.1 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Remarks:
(loading rate)
Basis for effect:
mortality
Remarks on result:
other: no visible/lethal effects in carp at the maximum soluble concentration in water generated by exposure of test media to a nominal loading rate of 100 mg/l (NOEC).
Details on results:
RANGE FINDING TEST:
The results of the range-finding test are presented in Table 1 (see any other information on results incl. tables section). No mortality or clinical effects were observed in any of the EA-3098 treated solutions.

Chemical analysis revealed that concentrations of all three peaks were below their limits of detection (see tables 4-6 of analytical report in attached background material).

All test conditions were maintained within the limits prescribed by the protocol.

FINAL LIMIT TEST:
Measured concentrations:
The results of analysis of the samples taken during the final test are shown in Tables 7-9 of the analytical report attached in background material.
Analysis of the samples again showed that measured concentrations of all three peaks were below their limits of detection.

Mortality and other effects:
Table 2 (see any other information on results incl. tables section) shows the mortality data recorded during the final test.
No effects were recorded in fish of either the control group or the EA-3098 treated solution.

Determination of effect concentrations:
Table 3 shows the effect parameters based on measured concentrations, i.e. the highest limit of detection of 0.15 mg/l.

Experimental conditions:
The results of measurement of pH and oxygen concentrations are presented in Table 4. The temperatures measured during the study in the various test vessels are presented in Table 5. All test conditions remained within the ranges prescribed by the protocol (pH: 6.0-8.5, constant
within 1 unit; temperature 20-24°C, constant within 2°C; oxygen> 60% of air saturation).











Results with reference substance (positive control):
Reference test:
96-hour acute toxicity study in the carp with pentachlorophenol (PCP).

The study procedure was as for the main study.

This reference test was carried out to check the sensitivity of the test system.

Concentrations: 0.10, 0.22 and 0.46 mg/l in ISO medium.

Incidence of mortality observed in the reference study:
Concentration PCP (mg/l) Initial number of fish Total mortality (%)
0.10 5 0
0.22 5 100 (3 dead at 24 hours, 5 dead at 48 hours)
0.46 5 100 (5 dead at 24 hours)

Under the conditions of this test PCP induced no lethal effects in carp at 0.10 mg/l. The 96h LC50 for carp exposed to PCP was 0.15 mg/l (95% confidence intervl between 0.10 and 0.22 mg/l) and was already reached within 48 hours of exposure.

The range of the 96 hour LC50 for carp is generally between 0.10 and 0.46 mg/l based on historical data of referencei item tests performed.

Table 1: Incidence of mortality and total mortality during the range-finding test

Concentration EA-3098 % of a 4.5 µm filtrate prepared at 100 mg/l

Initial number of fish

Cumulative mortality at time in hours

Total mortality

3

24

48

72

96

%

0.1

3

0

0

0

0

0

0

1.0

3

0

0

0

0

0

0

10

3

0

0

0

0

0

0

100

3

0

0

0

0

0

0

 

Table 2: Incidence of mortality and total mortality during the final test

Concentration EA-3098 % of a 4.5 µm filtrate prepared at 100 mg/l

Initial number of fish

Cumulative mortality at time in hours

Total mortality

3

24

48

72

96

%

0

7

0

0

0

0

0

0

100 (<0.15 mg/l)

7

0

0

0

0

0

0

Table 3: Effect parameters 

Parameter

Concentration EA-3098

NOEC

Solubility level (<0.15 mg/l)

24h LC50

>Solubility level (<0.15 mg/l)

48h LC50

>Solubility level (<0.15 mg/l)

72h LC50

>Solubility level (<0.15 mg/l)

96h LC50

>Solubility level (<0.15 mg/l)

Table 4: pH-values and dissolved oxygen concentrations (mg/l) during the final test

 

Concentration EA-3098 % of a 4.5 µm filtrate prepared at 100 mg/l

 

Day 0

Day 1

Day 2

Day 3

Day 4

pH

O2

pH

O2

pH

O2

pH

O2

pH

O2

0

7.8

9.3

7.6

7.5

7.6

7.2

7.4

7.1/7.9*

7.6

7.2

100

8.0

8.9

7.5

7.3

7.4

6.9

7.4

5.9/7.9*

7.5

7.1

*Aearation was applied for approximately 1.5 h, values are measured before and after aeration

Table 5: Temperatures (ºC) measured during the final test

 

Concentration EA-3098 % of a 4.5 µm filtrate prepared at 100 mg/l

 

Day 0

Day 1

Day 2

Day 3

Day 4

0

21.5*

21.8

21.8

21.9

21.8

100

21.7*

22.0

21.9

21.8

21.9

* Temperature of medium with acclimatised fish: 21.6ºC

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test EA-3098 induced no visible/lethal effects in carp at the maximum soluble concentration in water generated by exposure of test media to a nominal loading rate of 100 mg/l (NOEC).
The 96h LC50 exceeds the maximum solubility of EA-3098 in water, i.e. at a highest level of 1.0 x 10-4 g/l.
Executive summary:

96 -Hour Acute Toxicity Study in Carp with EA-3098

The study procedure described in this report was based on the ISO International Standard 7346-1: Static method, 1996. In addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C.1, 1992, the OECD guideline No. 203, 1992 and the OECD series on testing and assessment number 23, 2000.

 

The water solubility of EA-3098 was at the highest < 1.0 x 10-4 g/l (determined in a water solubility study). Preparation of test solutions started with weighed amounts of test substance that were quantitatively added to volumetric flasks. The test solutions were treated with ultra-sonic waves and stirred for 72 hours. Subsequently they were filtered through a 0.45 µm filter.

 

In a range-finding test no mortality or clinical effects were observed and a final limit test was performed with seven carp per test group exposed to a blank-control and a filtrate prepared at a loading rate of 100 mg/l. Samples were taken from the filtrates for chemical analyses during both the range-finding and final limit test. However, the levels of three major peaks remained below the limit of detection (< 0.15 mg/l). No effects were recorded in carp during the 96-hour period of exposure. The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

In conclusion: EA-3098 induced no visible/lethal effects in carp at the maximum solubility of EA3098 in water prepared at a nominal loading rate of 100 mg/l. The 96h-LC50 exceeds the maximum solubility of EA-3098 in water, i.e. at a highest level of 1.0 x 10-4 g/l.

Description of key information

Under the conditions of the test EA-3098 induced no visible/lethal effects in carp at the maximum soluble concentration in water generated by exposure of test media to a nominal loading rate of 100 mg/l (NOEC).

The 96h LC50 exceeds the maximum solubility of EA-3098 in water, i.e. at a highest level of 1.0 x 10-4 g/l.

Key value for chemical safety assessment

LC50 for freshwater fish:
0.1 mg/L

Additional information

96 -Hour Acute Toxicity Study in Carp with EA-3098

The study procedure described in this report was based on the ISO International Standard 7346-1: Static method, 1996. In addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C.1, 1992, the OECD guideline No. 203, 1992 and the OECD series on testing and assessment number 23, 2000.

 

The water solubility of EA-3098 was at the highest < 1.0 x 10-4g/l (determined in a water solubility study). Preparation of test solutions started with weighed amounts of test substance that were quantitatively added to volumetric flasks. The test solutions were treated with ultra-sonic waves and stirred for 72 hours. Subsequently they were filtered through a 0.45µmfilter.

 

In a range-finding test no mortality or clinical effects were observed and a final limit test was performed with seven carp per test group exposed to a blank-control and a filtrate prepared at a loading rate of 100 mg/l. Samples were taken from the filtrates for chemical analyses during both the range-finding and final limit test. However, the levels of three major peaks remained below the limit of detection (< 0.15 mg/l). No effects were recorded in carp during the 96-hour period of exposure. The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

In conclusion: EA-3098 induced no visible/lethal effects in carp at the maximum solubility of EA3098 in water prepared at a nominal loading rate of 100 mg/l. The 96h-LC50 exceeds the maximum solubility of EA-3098 in water, i.e. at a highest level of 1.0 x 10-4 g/l.