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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:

supporting study

Study period:

from March. 10 2008 to May 16 2008

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1, 5, 10, 20, 30, 40, 50, 80, 100 mg/L
- Sampling method: Samples were taken in duplicate from each concentration on four occasions during the definitive test. On each occasion, one sample was analysed at each concentration; the remaining samples was retained in case further analysis is required. The samples were flitted by 0.45 μm millipore filter, extracted with acetic-ester, then detected the content of
- Sample storage conditions before analysis:
- Concentrations:
- Sampling method: Samples were taken in duplicate from each concentration on four occasions during the definitive test. On each occasion, one sample was analysed at each concentration; the remaining samples was retained in case further analysis is required. The samples were flitted by 0.45 μm millipore filter, extracted with acetic-ester, then detected the content of
- Sample storage conditions before analysis:

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
- Eluate:
- Differential loading:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Test solutions were prepared with acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): amount of acetone < 0.1 % in test solution
- Evidence of undissolved material (e.g. precipitate, surface film, etc):

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Strain:
- Source: .Fishes were obtained from a commercial fish supplier of Guangzhou Hongfa Aquatic Animals Culture Company in China
- Age at study initiation (mean and range, SD):
- Length at study initiation (length definition, mean, range and SD): This study adopted the adult zebra fish (Brachydanio rerio) in accordance with the OECD test guidelines; the mean length of the batch of fish was between 1 and 3 cm.
- Weight at study initiation (mean and range, SD): 0;3 g
- Method of breeding:
- Feeding during test
- Food type:
- Amount:
- Frequency:

ACCLIMATION
- Acclimation period:
- Acclimation conditions (same as test or not):
- Type and amount of food:
- Feeding frequency:
- Health during acclimation (any mortality observed):

QUARANTINE (wild caught)
- Duration:
- Health/mortality:

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

140 mg CaCO3

Test temperature:

21 -22°C

pH:

7.18 to 7.93

Dissolved oxygen:

7.66 to 8.88 mgO2/L

Salinity:

not applicable

Nominal and measured concentrations:

According to the concentration levels determined by the range-finding test, nine concentration levels 1, 5, 10, 20, 30, 40, 50, 80, 100 mg/L) of the test substance were established. And also three treatment groups for each of the levels were used.

Details on test conditions:

TEST SYSTEM
- Test vessel: The prepared test dilutions were placed into the test vessels (glass aquaria) and gently aerated during the test. Each vessel was labelled with the study number, vessel number and the nominal exposure concentration.
For each test, five litres of different concentrations of test substance were applied.
The test fish were randomly assigned to the various treatment levels and controls with ten fish each. And maintain the following conditions:
-Light: 12 to 16 hours photoperiod daily;
-Temperature: 20 to 25℃ for zebra fish;

-Feeding: none.
-Test solution: renewed at 24 h with freshly made preparations.
The fish were inspected after 24, 48, 72 and 96 hours. Dead fish were removed when observed and mortalities were recorded. Observations at three and six hours after the start of the test were performed.
Meanwhile, measurement of pH, dissolved oxygen and temperature were carried out daily.
- Material, size, headspace, fill volume:
- Aeration: Oxygen concentration: at least 5.8 mg/L;
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel:10
- No. of vessels per concentration (replicates):
- No. of vessels per control (replicates):
- No. of vessels per vehicle control (replicates):
- Biomass loading rate:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine:
- Alkalinity:
- Ca/mg ratio:
- Conductivity:
- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod:
- Light intensity:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

K2Cr2O7 was used as the test substance

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

>= 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

12.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CI = 10.6-13.6 mg/L

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

30 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Results with reference substance (positive control):

- Results with reference substance valid?
- Mortality:
- LC50: 258 mg/L (24h)
- Other:

Validity criteria fulfilled:

yes

Conclusions:

This test is designed to determine the acute toxicity of test substances (DIMETHYL -2-METHYL GLUTARATE) to fish according to the following guidelines:
“The guidelines for the testing of chemicals”, SEPA (HJ/T 153-2004).
Procedure 203 of the “Guidelines for Testing of Chemicals” of the OECD: "Fish, Acute Toxicity Test” (1992).
The study met the acceptability criteria prescribed by the protocol and was considered valid.
The 96h LC50 of DIMETHYL -2-METHYL GLUTARATE to the zebra fish was 12.3 mg/l (95% confidence limits 10.6 to 13.6 mg/L). The highest concentration that was considered to have no treatment-related effects was 1.0 mg/L and the lowest concentration that caused 100% mortality after 96 hours was 30 mg/L.

Executive summary:

This test is designed to determine the acute toxicity of test substances(DIMETHYL -2-METHYL GLUTARATE)to fish according to the following guidelines:

“Theguidelinesfor the testing of chemicals”,SEPA(HJ/T 153-2004).

Procedure 203 of the“Guidelines for Testing of Chemicals”of the OECD: "Fish, Acute Toxicity Test” (1992).

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

The 96h LC₅₀of DIMETHYL -2-METHYL GLUTARATE to the zebra fish was 12.3 mg/l (95% confidence limits 10.6 to 13.6 mg/L). The highest concentration that was considered to have no treatment-related effectswas 1.0 mg/L and the lowest concentration that caused 100% mortality after 96 hours was 30 mg/L.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 29-oct-2007 to 11-jan-2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentrations of Dimethyl 2-methyl glutarate were measured in all test medium samples of the test concentrations of 22 to 100 mg/L. The samples taken from the two lowest test concentrations of 4.6 and 10 mg/L were not analyzed, since these test concentrations were below the 96-hour NOEC. From the control samples, one of the duplicate samples was analyzed from the corresponding sampling times.
- Sampling method: For the determination of the actual test item concentrations in this semi-static test with daily test medium renewal, duplicates samples were taken from the freshly prepared test media and the control at the start of the test (day 0) and at the last test medium renewal (day 3) and from the aged test media and the control at the end of the first and last test medium renewal period (days 1 and 4). However, no samples were taken from the highest nominal test concentration of 100 mg/L on days 3 and 4, since all fish were dead at this concentration by that time.
All samples were taken from the approximate center of the aquaria without mixing of the test medium.
- Sample storage conditions before analysis: All samples were deep-frozen immediately after sampling. The samples were stored at about -20°C until alalysis. The stability of the test item in samples under the storage conditions was confirmed in a pre-experiment (non-GLP).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
A concentrated stock solution of the nominal concentration of 500 mg/L was freshly prepared at the start of the test and before each test medium renewal by completely dissolving 1400 mg (dosed weight: 1400.4 mg) of test item in 2.8 L of test water or 3000 mg (dosed weight range: 2999.7-3000.2 mg) of test item in 6 L of test water unsing ultrasonic treatment for 10 min and stirring for 20 min at room temperature. Adequate volumes of the stock solution were added to the test water in the aquaria and were intensively mixed to prepare the test media with the test concentrations stated above. The test media were freshly prepared just before introduction of the fish at the start of the test and before each test medium renewal. The actual concentrations of the test item in the test media were analytically determined. The selection of the test concentrations was based on the results of a range-finding test (non-GLP).
- Controls: test water without test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Source: P. Hohler, trout breeding station Zeiningen (Switzerland)
- Age at study initiation (mean and range, SD): no data
- Length at study initiation (mean +/- SD): 5.6 +/- 0.15 cm
- Weight at study initiation (mean +/- SD): 1.53 +/- 0.18 g
- Feeding during test : no

ACCLIMATION
- Acclimation period: the fish were held in the laboratory for more than twelve days without any medication. Prior to the test start, they were acclimated for 1 week to the test water and temperature.
- Type and amount of food: commercial fish diet (Hokovit 502)
- Feeding frequency: until one day before the start of the test
- Health during acclimation (any mortality observed): During holding and acclimatization no fish died in the test fish batch and all fish were healthy

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

175 mg/L as CaCO3

Test temperature:

13-14°C

pH:

8.3-8.5

Dissolved oxygen:

Between 8.0 and 9.8 mg/L

Salinity:

not applicable

Nominal and measured concentrations:

Nominal concentration : 4.6, 10, 22, 46 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass aquarium with 15 L of test medium
- Aeration: The test water was aerated prior to the preparation of the test media until oxygen saturation was reached. During the test period, the test medium and the control were slightly aerated.
- Type of flow-through (e.g. peristaltic or proportional diluter): none (semi-static test)
- Renewal rate of test solution (frequency/flow rate): every day
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.7 g fish wet weight / L


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: local tap water (non chlorinated well water of drinking quality) reduced for total hardness by ion exchange
- Total organic carbon: no data
- Particulate matter: no data
- Metals: Cd < 0.5, As < 3, lead < 3, mercury < 1, selenium < 3, copper < 4 µg/L
- Pesticides: lindane, heptachlor, malathion, DDT total, dieldrin < 0.05 µg/L
- Chlorine: no data
- Alkalinity: no data
- Ca/mg ratio: no data
- Conductivity: no data
- Bacteriological parameters: at the time of sampling, the tested parameters met the requirement for drinking water.
- Culture medium different from test medium: no data
- Intervals of water quality measurement: The water temperature, pH values and oxygen concentrations were measured in each test concentration with surviving fish and in the control at the start of the test and once every day during the test in the freshly prepared and old test medium. At the same dates the appearance of the test medium was recorded. The total hardness of the water used in the test was measured at the start of the test in the control.


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16-hour light and 8-hour darkness with a 30-minute transition period
- Light intensity: 50- 500 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The test fish were observed after approximately 3, 24, 48, 72 and 96 hours test duration for mortality and visible abnormalities. Dead fish were removed at least once daily and discarded.
The 96-hour LC50 and its 95% confidence interval were calculated by Probit Analysis. The NOEC, LOEC, LC0, and LC100 were determined directly from the raw data.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study : yes
- Test concentrations: loading rate of 100 mg/L
- Results used to determine the conditions for the definitive study: no mortality at the undiluted loading rate.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

ANALYTICAL MONITORING
The analytically determined concentrations of the test item in the test media of the nominal test concentration of 22 to 100 mg/L were between 83 and 94% of the nominal values at the start and the end of the test medium renewal periods. Thus, the test item was stable during the test medium renewal period of 24 hours. The biological results are based on the nominal concentration of the test item.

BIOLOGICAL RESULTS
In the control and at the test concentrations up to and including 22 mg/L, all fish survived until the end of the test and no visible abnormalities were observed. At the next higher test concentration of 46 mg/L, all test fish showed symptoms of toxicity after 48 hours of exposure and one fish died during the test. At the highest test concentration of 100 mg/L, all fish were dead already after 48 hours of test duration.

APPEARANCE OF THE TEST MEDIUM
No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test medium renewal periods.

Results with reference substance (positive control):

not tested

Reported statistics and error estimates:

not necessary

Sublethal observations / clinical signs:

Mortality and visible abnormalities observed in the test fish :

Number of fish tested at each concentration and the control: 7

Nominal test item concentration (mg/L)	Number of abnormal and dead fish / number of dead fish Type of visible abnormalities
	Observation time
	3 hours	24 hours	48 hours	72 hours	96 hours
Control	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
4.6 10 22 46     100	0 / 0 0 / 0 0 / 0 0 / 0     0 / 0	0 / 0 0 / 0 0 / 0 0 / 0     7 / 6 AP, BO	0 / 0 0 / 0 0 / 0 7 / 0 AP, TS   7 / 7	0 / 0 0 / 0 0 / 0 7 / 0 AP, TS, BO, VF - / -	0 / 0 0 / 0 0 / 0 7 / 1 AP, TS, BO, VF - / -
LC50 95% C.I.	> 100 -	72 n.d.	68 46-100	68 46-100	56 42-74

 

- / - : All fish dead

95% C.I.: 95% confidence interval of the LC50

n.d.: could not be determined

 

AP: Apathy

BO: Fish mainly at the bottom of the aquarium

TS: Tumbling during swimminig

VF: Changed body color

Validity criteria fulfilled:

yes

Conclusions:

No mortality was observed up to 22 mg/l. As the 96-hour LC50 was calculated to be 56 mg/l, DIMETHYL 2-METHYL GLUTARATE is considered as harmful to the tested aquatic organisms.

Executive summary:

The acute toxicity of the test item Dimethyl 2 -methyl glutarate to rainbow trout (Oncorhynchus mykiss) was determined in a 96-hour semi-static test with a daily test medium renewal according to the EU Commission Directive 92/69/EEC, Part C.1 (1992), and the OECD Guideline for Testing of Chemicals No. 203 (1992).

Rainbow trouts were exposed to control and test chemical at the nominal concentrations of 4.6, 10, 22, 46, and 100 mg/L. The test fish were observed after approximately 3, 24, 48, 72 and 96 hours test duration for mortality and visible abnormalities.

In the control and at the test concentrations up to and including 22 mg/L, all fish survived until the end of the test and no visible abnormalities were observed. At the next higher test concentration of 46 mg/L, all test fish showed symptoms of toxicity after 48 hours of exposure and one fish died during the test. At the highest test concentration of 100 mg/L, all fish were dead already after 48 hours of test duration.

Thus, the 96-hour NOEC and the 96-hour LC0 of Dimethyl 2 -methyl glutarate to rainbow trout were both determined to be 22 mg/L. The 96-hour LOEC was 46 mg/L. The 96-hour LC50 of Dimethyl 2 -methyl glutarate was calculated to be 56 mg/L with a 95% confidence interval from 42 to 74 mg/L. The 96-hour LC100 was 100mg/L.

As the 96-hour LC50 was calculated to be 56 mg/l, DIMETHYL 2-METHYL GLUTARATE is considered as harmful to the tested aquatic organisms.

Description of key information

The acute toxicity to fish of Dimethyl 2-methyl glutarate was investigated in two studies performed according to the OECD Guideline for Testing of Chemicals No. 203 (1992). Two fish species were tested, the zebra fish (Danio rerio) and the rainbow trout (Oncorhynchus mykiss); 96-hour LC50 values of 12.3 mg/L and 56 mg/L were reported, respectively. The lowest value was used for the assessment.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

12.3 mg/L

Additional information

Two experimental studies, scored as Klimisch 1, are available (Shi L., 2008; Bätscher R., 2008) and selected as a key studies.