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Ecotoxicological information

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:
2010-02-01-2010-02-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in accordance with OECD No. 203 without deviation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
-
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.10, 0.18, 0.32, 0.56 and 1.0 mg/l
- Sampling method: 2 ml from the approximate center of the test vessel
- Frequency: at the start of the test and after 72 hours from the freshly prepared solutions; at the first renewal (t=24h) and the end of the test from the 24-hour old solutions
- Sample storage conditions before analysis: not applicable, samples were analysed on the day of sampling.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: preparation of test solutions started with stock solutions in acetone at a factor of 10,000 higher than the final test concentrations. Volumes of 100 µl of the respective stocks were added per litre of test medium and stirred one day in a closed vessel with minimal headspace. The final test solutions were all clear and colourless.
- Eluate:
- Differential loading: 0.32 g fish/litre, i.e. 7 fish per 6 litres
- Controls: test medium without test substance but containing acetone as used for the stock solutions (solvent control)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no undissolved material
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: Carp
- Strain: Cypirinus carpio, Teleostei, Cyprinidae
- Source: Zodiac, proefacc, 'De Haar Vissen', Wageningen University and Research Centre, The Netherlands
- Age at study initiation:
- Length at study initiation: 2.2 ± 0.1 cm
- Weight at study initiation: 0.27 ± 0.04g
- Method of breeding: F1 from a single parent-bred in UV-treated water
- Feeding during test: no feeding from 24 hours prior to the test and during the total test period

ACCLIMATION
- Acclimation period: at least 12 days after delivery
- Acclimation conditions (same as test or not): yes
- Type and amount of food: pelleted fish food
- Feeding frequency: daily
- Health during acclimation (any mortality observed): 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%

QUARANTINE (wild caught)
Not applicable
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None. The surviving fish were killed at the end of the test.
Hardness:
180 mg CaCO3.
Test temperature:
21°C.
pH:
7.
Dissolved oxygen:
Between 6-9.0 mg/l
Salinity:
-
Nominal and measured concentrations:
See table 1.
Details on test conditions:
TEST SYSTEM
- Test vessel: 6.5 litres, all-glass, containing 6 litres of test solution
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: minimal headspace
- Aeration: the test medium was not aerated during the test
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 7 fish per concentration
- No. of vessels per concentration (replicates):
- No. of vessels per control (replicates):
- No. of vessels per vehicle control (replicates):

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands)
- Culture medium different from test medium:
- Intervals of water quality measurement: pH, nitrate and ammonia concentration: once a week.
- Temperature: every day. In addition, pH and temperature were measured before transferring the fish to the test system.

OTHER TEST CONDITIONS
- Adjustment of pH: not necessary
- Photoperiod: 16 hours photoperiod daily
- Light intensity: at low light intensity

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.1, 1.0 and 10 mg/l
- Results used to determine the conditions for the definitive study: yes. In the range-finding test there was already 100% mortality at 1.0 mg/l, therefore the concentrations used in the definitive test did not surpass 1.0 mg/l.
Reference substance (positive control):
yes
Remarks:
Pentachlorophenol (PCP)
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
0.21 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: 0.16-0.28 mg/l
Details on results:
- Behavioural abnormalities: none
- Other biological observations: clinical effects were limited to effects on swimming behaviour
- Mortality of control: no
- Other adverse effects control: none
- Abnormal responses: -
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no, solubility of mmt was 10 mg/l
Results with reference substance (positive control):
- Results with reference substance valid? Yes, the LC50 for carp exposed to PCP was within the range of 0.10 and 0.46 mg/l, values which are based on the historical data of NOTOX
- Mortality: yes, 100% mortality at the second concentration tested (0.22 mg/l)
- LC50: 0.15 mg/l C.I. 0.10-0.22 mg/l
Reported statistics and error estimates:
The LC50 could not be determined using the maximum likehood estimation method with the probits of the percentages of dead fish as function of the logarithms of the corresponding concentrations. This was because there was no concentration between the highest concentration (A) at which 100% mortality and the lowest concentration (B) at which 0% mortality occurred. Instead, the LC50 was calculated as (AB)^1/2, with A and B being limits of the 95% C.I.
Sublethal observations / clinical signs:

Table 2 – Incidence of mortality and total mortality during the range-finding test

Concentration

mmt (mg/l)

Initial number

of fish

Cumulative mortality

Total mortality

(%)

21/4h

24h

48h

72h

96h

0.1

3

0

0

0

0

0

0

1.0

3

0*

3

3

3

3

100

10

3

3

3

3

3

3

100

*Fish were all dead at the end of the day (~6hours)

Table 3 – Incidence of mortality and total mortality during the final test

Concentration

mmt (mg/l)

Initial number

of fish

Cumulative mortality

Total mortality

(%)

33/4h

24h

48h

72h

96h

Control

7

0

0

0

0

0

0

0.09

7

0

0

0

0

0

0

0.16

7

0

0

0

0

0

0

0.28

7

2

7

7

7

7

100

0.49

7

5

7

7

7

7

100

0.81

7

5

5

6

7*

7

100

*The last fish was euthanized for humane reasons and treated as having died in the test based on the Dutch Act on Animal Experimentation (Feb.1997) and the OECD guideline on humane endpoints (ENV/JM/MOONO/(2000)7).

Table 4 – Clinical effects observed during the final test

Concentration

mmt (mg/l)

Time of recording

(hours)

Specification of effects

Relative

number

Control, 0.09

0-96

No abnormalities

7/7

0.16

3 ¾

Swimming at the surface with loss of righting reflex

7/7

0.28

3 ¾

Swimming at the surface with loss of righting reflex

5/5

0.49

3 ¾

Swimming at the surface with loss of righting reflex

2/2

0.81

3 ¾-24

48

72

Swimming at the surface with loss of righting reflex

Swimming at the surface with loss of righting reflex

Swimming at the surface with loss of righting reflex

2/2

1/1

1/1

 

Table 5 – Effect parameters

Parameter

Concentration mmt

(mg/l)

95%- C.I.

(mg/l)

LC0

0.16

-

24, 48, 72, 96h – LC50

0.21

0.16-0.28

Validity criteria fulfilled:
yes
Remarks:
The negative and positve controls provided expected results. Test conditions were maintained constant throughout the test. The dissolved oxygen concentration was at least 60% of the air saturation value throughout the test (>5mg/l at 22°C)
Conclusions:
Under the conditions of the study, mmt induced no lethal effects in carp at or below an average exposure concentration of 0.16 mg/l (LC0).
The 96h-LC50 was assessed to be 0.21 mg/l based on average exposure concentrations (95% C.I. between 0.16 and 0.28 mg/l).
Executive summary:

The study protocol was based on the OECD Guideline 203 (1992). The protocol was designed to meet the specifications of the EC/440/2008, Part C. 1, 2008, ISO International Standard 7346-2: Semi-static method (19960 and the OECD series on testing and assessment number 23 (2000).

The batch of mmt tested was a clear yellow-orange liquid with a purity >96%. The test material was completely soluble in test medium at the concentrations tested.

Stock solutions in acetone at a factor of 10,000 higher than the final test concentrations were used for spiking. A subsequent stirring period of one day resulted in completely dissolved clear and colorless test solutions.

A definitive test was performed based on the results of a preceding range-finding test. Seven fish per test group were exposed to a control and a nominal mmt concentration range between 0.10 and 1.0 mg/l with a spacing factor of 1.8. The total test period was 96 hours and test solutions were renewed on a daily basis. Samples for analytical confirmation of actual exposure concentrations were taken from the freshly prepared solutions at the start and 72 hours of exposure and from 24-hour old solutions at 24 and 96 hours of exposure.

Analysis of the samples taken from the freshly prepared solutions (t=0 and t=72h) showed that measured concentrations were in agreement with nominal (recoveries 95-109% relative to nominal). The measured concentrations in the 24-hour old solutions decreased on average by 20 to 30% below initial. The average exposure concentrations were calculated to correspond to 0.09, 0.16, 0.28, 0.49 and 0.81 mg/l at the nominal concentrations of respectively 0.10, 0.18, 0.32, 0.56 and 1.0 mg/l.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

 

mmt induced no lethal effects in carp at or below an average exposure concentration of 0.16 mg/l (LC0). The 96h-LC50 of 0.21 mg/l was based on average exposure concentrations (95% C.I. between 0.16 and 0.28 mg/l). The result was reached after 24 hours of exposure.

Description of key information

The 96h-LC50 of 0.21 mg/l was based on average exposure concentrations (95% C.I. between 0.16 and 0.28 mg/l). The result was reached after 24 hours of exposure.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
0.21 mg/L

Additional information

mmt induced no lethal effects in carp at or below an average exposure concentration of 0.16 mg/l (LC0).