4-Decenal, 9-hydroxy-5,9-dimethyl-

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:

From 2018-11-20 to 2019-04-18

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:

other: GB/T 29763-2013 Chemicals-Rare Minnow (Gobiocypris rarus) acute toxicity test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Signed on 2019/04/18

Analytical monitoring:

yes

Details on sampling:

Measurements of the pH, dissolved oxygen concentration and temperature in the test solutions were made at the beginning, before and after each renewal, and at the end of the test.
For the analysis of the actual test substance concentration, 30 ml of test solution was taken from each test vessel at the beginning, before and after each renewal, and at the end of the test.

Vehicle:

no

Details on test solutions:

2.0004 g test substance was dissolved in 2 L test water to prepare a stock solution with a nominal concentration of 1g/L. The test solutions were prepared by dilution of the stock solution with test water. Based on the results of the range-finding test, five treatment groups and a contrai group were included in the definitive test. The nominal concentrations of the treatment groups were 10 mg/L, 14 mg/L, 20 mg/L, 28 mg/Land 40 mg/L, the geometric mean measured concentrations of which were 9.53 mg/L, 12.8 mg/L, 19.3 mg/L, 28.6 mg/L and 40.0 mg/L, repectively.

Test organisms (species):

other: Gobiocypris rarus

Details on test organisms:

The test was performed with Gobiocypris rarus which were bred in Bioassay and Safety Assessment Lab, Shanghai Academy of Public Measurement (ID:GR-SAPM-180515). This freshwater fish species is suitable for fish toxicity testing because of their ease of culturing and handling, sensitivity to a variety of chemical substances, and the extensive data base for this species. This species complies with "Ministry of environmental protection of the People's Republic of China, The Guidelines for the Testing of Chemicals-Effects on Biotic System, 2013, 203 Fish, Acute Toxicity Test", "OECD, Guidelines for the Testing of Chemicals. 203 Fish, Acute Toxicity Test, 1992" and "GB/T 29763-2013 Chemicals-Rare Minnow (Gobiocypris rarus) acute toxicity test".
Before being used in the test, all fish were acclimatized in the following test conditions for more than 14 days:
Water: the _test water used was tap water dechlorinated by activated carbon and sterilized by UV lamp;
Temperature: 21-25°C and constant within a range of 2°C.
pH: 6.0-8.5;
Dissolved oxygen concentration: > 80% of air saturation value (ASV);
Photoperiod: Light/Dark = 12 hours/12 hours;
Feeding: fish were fed with brine shrimp flake (O.S.I®, USA) daily until 24 hours before the test was started.
Length was 2.2 ± 0.1 cm (Mean ± SD) and weight was 0.17 ± 0.02 g (Mean ± SD) at the end of the test.
No diseases were observed during the acclimation period;
No mortality was observed within the 7 days before the start of the test.
No feeding during test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

Test media was renewed every 24h during the definitive test.

Post exposure observation period:

None.

Hardness:

No data.

Test temperature:

21.0 - 21.9°C

pH:

No data.

Dissolved oxygen:

79.8%-94.2%ASV

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Nominal concentrations: 10 mg/L, 14 mg/L, 20 mg/L, 28 mg/L and 40 mg/L
Geometric mean measured concentrations: 9.53 mg/L, 12.8 mg/L, 19.3 mg/L, 28.6 mg/Land 40.0 mg/L

Details on test conditions:

Water: Tap water, dechlorinated by activated carbon and sterilized by UV lamp, was used. The water was aerated continuously over 24 h.
Test vessel: Five litre glass tanks were used containing 4 L test solution.
Loading: 7 fish/ test vessel
Photoperiod: Light/Dark=12 hours/12 hours

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 40 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other:

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

35.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%CL: 30.7 - 40.5 mg/L

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

28.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%CL: 25.2 - 33.2 mg/L

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

24.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%CL: 22.5 - 27.3 mg/L

Details on results:

ln the definitive test, the maximum mean measured concentration causing no mortality and the minimum mean measured concentration causing 100 percent mortality within the period of the test was 19.3 mg/L and 40.0 mg/L, respectively.There were no mortalities or abnormalities observed in fish of the control group during the test.

Results with reference substance (positive control):

For evaluation of the fish quality and experimental conditions, potassium dichromate was tested as a positive contrai to demonstrate satisfactory test conditions once a year (recently performed on 2018.6.4-6.8). The 96 h LC50 value of the test substance to Gobiocypris rarus (ID: GR-SAPM-180112) was 257 mg/L (95% confidence limits: 207-309 mg/L), which was within the ± 2 SD range of the QC chart in our lab (82.2-310 mg/L).

Reported statistics and error estimates:

The mortalities were plotted against each test concentration (geometric mean measured) and control for each exposure period (24 h, 48 h, 72 h and 96 h). The 24, 48, 72 and 96 hours LC50 with 95% confidence limits were calculated using the method of Spearman-Karber. The 24 hours LC50 were calculated using the method of Log-Logit Interpolation. The data analysis was carried out with software TOXCALC-Toxicity Data Analysis Software (v5.0.32).

Sublethal observations / clinical signs:

The mortalities and abnormalities in first range-finding test (5 fish in each vessel)

Group		Duration (h)	Mortalities	Cumulative Mortalities	Abnormalities
Blank Control		0	0	0	none
		24	0	0	none
		48	0	0	none
		72	0	0	none
		96	0	0	none
Treatment Group (mg/L)	12	0	0	0	none
		24	0	0	none
		48	0	0	none
		72	0	0	none
		96	0	0	none
	60	0	0	0	none
		24	5	5	/
		48	/	5	/
		72	/	5	/
		96	/	5	/
	120	0	0	0	none
		24	5	5	/
		48	/	5	/
		72	/	5	/
		96	/	5	/
/, no surviving fish .

The mortalities and abnormalities in first range-finding test (7 fish in each vessel)

Group		Duration (h)	Mortalities	Cumulative Mortalities	Abnormalities
Blank Control		0	0	0	none
		24	0	0	none
		48	0	0	none
		72	0	0	none
		96	0	0	none
Treatment Group (mg/L)	10	0	0	0	none
		24	0	0	none
		48	0	0	none
		72	0	0	none
		96	·o	0	none
	16	0	0	0	none
		24	0	0	none
		48	0	0	none
		72	0	0	none
		96	0	0	none
	25	0	0	0	none
		24	0	0	none
		48	0	0	none
		72	0	0	One lost equilibrium
		96	2	2	One lost equilibrium
	39	0	0	0	none
		24	0	0	none
		48	3	3	none
		72	4	7	/
		96	/	7	/
	61	0	0	0	none
		24	6	6	One lost equilibrium
		48	1	7	/
		72	/	/	/
		96	/	/	/
/, no surviving

The mortalities and abnormalities in definitive test (7 fish in each vessel)

Group		Duration (h)	Mortalities	Cumulative Mortalities	Abnormalities
Blank Control		0	0	0	none
		3	0	0	none
		24	0	0	none
		48	0	0	none
		72	0	0	none
		96	0	0	none
Treatment Group (mg/L)	9.53	0	0	0	none
		3	0	0	none
		24	0	0	none
		48	0	0	none
		72	0	0	none
		96	0	0	none
	12.8	0	0	0	none
		3	0	0	none
		24	0	0	none
		48	0	0	none
		72	0	0	none
		96	0	0	none
	19.3	0	0	0	none
		3	0	0	none
		24	0	0	none
		48	0	0	none
		72	0	0	none
		96	0	0	none
	28.6	0	0	0	none
		3	0	0	none
		24	0	0	none
		48	1	1	none
		72	2	3	none
		96	3	6	none
	40	0	0	0	none
		3	0	0	none
		24	1	1	none
		48	4	5	none
		72	2	7	/
		96	/	7	/

Validity criteria fulfilled:

yes

Conclusions:

The 96 h LC50 value for the test substance to Gobiocypris rarus was 24.8 mg/L and its 95% confidence limits ranged from 22.5 to 27.3 mg/L. The maximum mean measured concentration causing no mortality and the minimum mean measured concentration
causing 100 percent mortality within the· period of the test were 19.3 mg/L and 40 mg/L, respectively.

Executive summary:

The acute toxicity of the test substance to Gobiocypris rarus was determined during a semi-static test under GLP conditions. This test was performed according to "Ministry of environmental protection of the People's Republic of China, The Guidelines for the Testing of Chemicals-Effects on Biotic System, 2013, 203 Fish, Acute Toxicity Test", "OECD, Guidelines for the Testing of Chemicals. 203 Fish, AcuteToxicity Test, 1992" and "GB/T 29763-2013 Chemicals-Rare Minnow (Gobiocypris rarus) acute toxicity test",

2.0004 g test substance was dissolved in 2 L test water to prepare a stock solution with a nominal concentration of 1 g/L. The test solutions were prepared by dilution of the stock solution with test water. Based on the results of the range-finding test, five treatment groups and a contrai group were included in the definitive test. The nominal concentrations of the treatment groups were 10 mg/L, 14 mg/L, 20 mg/L, 28 mg/Land 40 mg/L, the geometric mean measured concentrations of which were 9.53 mg/L, 12.8 mg/L, 19.3 mg/L, 28.6 mg/L and 40.0 mg/L, repectively. The test duration was 96 h and the frequency of renewal in the definitive test was every 24 h for test solutions and the stock solution was not renewed during the definitive test.

No mortality occurred in the contrai during the test and the dissolved oxygen concentration was greater than 60.0% of the air saturation value (ASV) throughout the test duration. Hence, the test was considered to be valid.

During the test, the variation of the concentration analyzed by Gas Chromatography (GC) was greater than 20% (-21.6%) only in case of the lowest treatment group at 72 h renewal, while the others all varied less than 20% during each renewal period. The freshly prepared solutions varied within ± 20% of their arithmetic mean measured concentration (-18.1 % to 12.9%).

The 96 h LC50 value for the test substance to Gobiocypris rarus was 24.8 mg/L and its 95% confidence limits ranged from 22.5 to 27.3 mg/L. The maximum mean measured concentration causing no mortality and the minimum mean measured concentration causing 100 percent mortality within the· period of the test were 19.3 mg/L and 40 mg/L, respectively. The results above were expressed relative to the geometric means of the measured concentrations.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

08 July to 14 August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Storage condition of test material: Protect from light, refrigerator.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 2.2, 4.6, 10, 22, 46, 100 mg/L
- Sampling method: For the determination of the actual test item concentrations in this semi-static test, duplicate samples were taken from each treatment just before the start of the first and of the last (forth) renewal periods (Days 0 and 3). At the end of these renewal periods (Days 1 and 4), samples were taken for determination of the stability of the test item during the renewal periods of 24 hours. Additionally, fresh and aged samples were taken from 4.6 and 22 mg/L from the second and third renewal period. From the highest nominal test concentration of 100 mg/L, the last samples were taken after 24 hours.
- Sample storage conditions before analysis: All samples were stored deep-frozen (at about -20 °C) immediately after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A stock solution of nominal 200 mg/L was freshly prepared by completely dissolving 1000 mg of the test item (dosed in the range of 1.0002 to 1.0044 g) in 5000 mL of test water using stirring for 20 minutes at room temperature. Then, adequate volumes of the intensively stirred stock solution were diluted with test water to obtain the desired test concentrations. The test media were freshly prepared just before introduction of the fish (= start of the exposure) and before each test medium renewal.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebra fish
- Strain: Danio rerio
- Source: obtained from a breeding culture at Harlan Laboratories.
- Length at study initiation (measured from the mouth to the end of the tail fin; mean ± SD): 3.0 ± 0.14
- Weight at study initiation (mean ± SD): 0.20 ± 0.03 g

ACCLIMATION
- Acclimation period: 1 week
- Acclimation conditions: same as test
- Type and amount of food during acclimation: A commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, 49304 Melle / Germany).
- Health during acclimation (any mortality observed): No mortality and all fish were healthy

FEEDING DURING TEST: Not fed

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

125 mg/L as CaCO3

Test temperature:

21-22 °C

pH:

6.9-7.1

Dissolved oxygen:

8.2-8.8 mg/L

Nominal and measured concentrations:

Nominal concentration: 4.6, 10, 22, 46 and 100 mg/L
Measured concentration: 5.2, 10.8, 23.9, 52.6 and 118 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: One glass test vessel (length: 24 cm; width: 17 cm, height: 21 cm; height of water level: 13 cm)
- Aeration: slightly aerated
- Renewal rate of test solution (frequency): daily
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.28 g fish wet weight per liter test medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Reconstituted test water was used in the study. It consisted of analytical grade salts dissolved in purified water to obtain the following nominal concentrations:

CaCl2 × 2H2O: 147 mg/L
MgSO4 × 7H2O: 61.5 mg/L
NaHCO3: 32.5 mg/L
KCl: 2.9 mg/L
Water Hardness: 125 mg/L as CaCO3 mg/L
Alkalinity: 0.4 mmol/L
The ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity. The test water was aerated prior to the preparation of the test media until oxygen saturation was reached.

- Intervals of water quality measurement: The water temperature, pH values and oxygen concentrations were measured for each treatment with surviving fish at the start of the test and once daily during the test in the freshly prepared and old test media.

OTHER TEST CONDITIONS
- Adjustment of pH: Not adjusted
- Photoperiod: 16-hour light to 8-hour dark photoperiod
- Light intensity: 140 to 480 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortality and Visible Abnormalities, pH, Oxygen Concentrations

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: 3 of 3 fish were dead at the concentration of 100 mg/L.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

41 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CI

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

46 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CI

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

24 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

118 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

24 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

not specified

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

53 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

not specified

Results with reference substance (positive control):

Not Applicable

Sublethal observations / clinical signs:

In the analysed test medium samples, the measured test item concentrations ranged between 100 and 118% of the nominal values.

Consequently, the test water renewal ensured nearly constant concentration of the test item during the test. The reported biological results are related to both the nominal and the following mean measured test item concentrations:

Table 6.1.1/3: The concentration of test item measured

Nominal test concentration (mg/L)	Mean measured concentration of the test item (geometric mean)
	(mg/L)	% of nominal
4.6	5.2	112
10	10.8	108
22	23.9	109
46	52.6	114
100	118	118

 

The biological results (visible abnormalities observed at the test fish, mortality and the LC50 values) are listed in Table 1. 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 in the test fish. At the next higher test concentration of 46 mg/L, all test fish showed one or several visible abnormalities. At the end of the test, five of the seven test fish died at this test concentration. At the highest test concentration of 100 mg/L, all fish were dead already after 24 hours of test duration.

Thus, the 96-hour NOEC and LC0 of GR-87-6984-4 to zebra fish were both determined to be 22 mg/L (nominal) and 24 mg/L (mean measured). The 96-hour LOEC was 46 mg/L (nominal) and 53 mg/L (mean measured).

The 96-hour LC50 of GR-87-6984-4 was calculated to be 41 mg/L with a 95% confidence interval of 34-49 mg/L (nominal) and 46 mg/L with a 95% confidence interval of 34-62 mg/L (mean measured). The 96-hour LC100 was 100 mg/L (nominal) and 118 mg/L (mean measured).

No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the entire test duration. The pH values in all treatments ranged from 6.9 to 7.1. The oxygen concentration was at least 8.2 mg/L, corresponding to at least 91% oxygen saturation. The water temperature was 21-22 °C during the test.

The test was considered to be valid, since no mortality in the control was observed and the validity criterion of at least 60% oxygen saturation was fulfilled.

Validity criteria fulfilled:

yes

Conclusions:

The test item had acute toxic effects on juvenile zebra fish (Danio rerio). The biological test results after 96 hours exposure based on both nominal and mean measured exposure concentrations were as follows:
Based on nominal (mg/L);Based on mean measured (mg/L)
96-hour LC50: 41;46
95% confidence interval: 34-49;34-62
96-hour LC0: 22;24
96-hour LC100: 100;118
96-hour NOEC: 22;24
96-hour LOEC: 46;53

Executive summary:

The acute toxicity of the test item to zebra fish (Danio rerio) was determined in a 96-hour semi-static test with daily test medium renewal. The test was performed in accordance with OECD Guideline 203 (Fish, Acute Toxicity Test), EU Method C.1 (Acute Toxicity for Fish) and EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test).

Test item was tested at the concentrations of 4.6, 10, 22, 46 and 100 mg/L along with a control group. Test medium samples were analysed and the measured test item concentrations ranged between 100 and 118% of the nominal values.

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 in the test fish. At the next higher test concentration of 46 mg/L, all test fish showed one or several visible abnormalities. At the end of the test, five of the seven test fish died at this test concentration. At the highest test concentration of 100 mg/L, all fish were dead already after 24 hours of test duration.

The biological test results after 96 hours exposure based on both nominal and mean measured exposure concentrations were as follows:

 

Table 6.1.1/4: The Results of the Acute Toxicity for Fish

	Based on nominal (mg/L)	Based on mean measured (mg/L)
96-hour LC50:	41	46
95% confidence interval:	34-49	34-62
96-hour LC0:	22	24
96-hour LC100:	100	118
96-hour NOEC:	22	24
96-hour LOEC:	46	53

 

The test was considered to be valid, since no mortality in the control was observed and the validity criterion of at least 60% oxygen saturation was fulfilled.

 

Description of key information

Two acute toxicity tests were conducted on the registered substance. Both OECD 203 studies (semi-static) were performed under GLP conditions and fulfill the validity criteria.

In a first study (Harlan, 2013), the acute toxicity of the test item to zebra fish (Danio rerio) was determined after 96 hour, a LC50 of 46 mg/L was found.

In a second study (Shanghai Academy of Public Measurement, 2019), the acute toxicity of the test item to Chinese rare Minnow (Gobiocypris rarus) was determined after 96 hour, a LC50 of 24.8 mg/L was found.

The lowest LC50 value is chosen as endpoint for acute toxicity on fish.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

24.8 mg/L

Additional information

To assess the acute toxicity of the substance to fish, two studies are available, in accordance to international guidelines with GLP statement.

 

The first study, performed by Harlan laboratory is considered valid. Following a preliminary range-finding test, zebra fish were exposed to aqueous solutions of the test material over a range of nominal concentrations of 4.6, 10, 22, 46 and 100 mg/L for a period of 96 -h, under semi-static test conditions. Chemical analysis of test samples was performed using GC-FID. In the analyzed test medium samples the measured test item concentrations ranged between 100 and 118% of the nominal values.Consequently, the test water renewal ensured nearly constant concentration of the test item during the test. In the control and at the test concentrations up to and including 22 mg/L, all fish survived untilthe end of the test and no visible abnormalities were observed in the test fish. At the next highertest concentration of 46mg/L, all test fish showed one or several visible abnormalities. At theend of the test, five of the seven test fish died at this test concentration. At the highest test concentration of 100 mg/L, all fish were dead already after 24 hours of test duration.

The LC50(96h), based on the mean measured test concentrations of the test media, was 46 mg/L with 95% confidence limits of 34 -62 mg/L. The highest measured concentration without observed effect was 24 mg/L.

 

The second study, performed by the laboratory of Shanghai Academy of Public Measurement is also considered as valid.

Chinise rare Minnow fish were exposed to aqueous solutions of the test material over a range of nominal concentrations of 10, 14, 20, 28 and 40 mg/L for a period of 96 -h, under semi-static test conditions. Fish were observed after 0, 24, 48, 72 and 96h.

No mortality occurred in the contrai during the test and the dissolved oxygen concentration was greater than 60.0% of the air saturation value (ASV) throughout the test duration. Hence, the test was considered to be valid.

During the test, the variation of the concentration analyzed by Gas Chromatography (GC) was greater than 20% (-21.6%) only in case of the lowest treatment group at 72 h renewal, while the others all varied less than 20% during each renewal period. The freshly prepared solutions varied within ± 20% of their arithmetic mean measured concentration (-18.1 % to 12.9%).

The 96h-LC50 value for the test susbstance was 24.8 mg/L based on measured concentrations, with a 95% confidence limits ranging from 22.5 to 27.3 mg/L.

Both studies are considered as being relevant and valid. The lowest 96h-LC50 value is selected as key value for acute toxicity to fish.