1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

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:

22 May 1982 to 26 May 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

not GLP compliant

Qualifier:

no guideline followed

Principles of method if other than guideline:

Method was performed in accordance with the test facility's Test Protocol for Fishes: 'Static, Acute Marine Fish Toxicity Test' dated February 1980.

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

A 100 mL water sample for chemical analysis was taken from each treatment (25 mL from each of the replicates) at the initiation (day 0) of the test and on test days 2 and 4.

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

A stock solution was prepared by mixing 1.6537 g of the test substance in 100 mL of reagent grade acetone. For each test concentration, an appropriate volume of stock solution was pipetted into the seawater in the test jars. A solvent control and a seawater control were maintained concurrently. Each solvent control jar received a volume of acetone equivalent to the greatest volume added to the test jars (5 mL), but no test material. The seawater control received no test material or acetone.

Test organisms (species):

Leiostomus xanthurus

Details on test organisms:

- Common name: Spot
- Source: Juvenile Leiostomus xanthurus were collected from Big Lagoon, a Gulf of Mexico estuary adjacent to the test facility
- Acclimation period: 38 days
- Holding condition: the salinity was 27 - 28 ‰ and the seawater temperature was 25 - 26°C.
- Mortality was < 1% during the 48-hour period immediately before testing.
- Weight of control fish at study initiation: 0.37 - 1.58 g wet weight; mean weight was 0.80 (S.D.±0.36) g
- Length of control fish at study initiation: 29 - 44 mm; mean length was 35 (S.D.+4) mm
The control fish appeared to be representative of the population.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

22 - 23 °C

pH:

- Test initation: 8.1 (in all treatments)
- Test termination: 7.4 - 8.0 (in all treatments containing live fish)

Dissolved oxygen:

- Test initation: ≥ 92% of saturation
- Test termination: ≥ 74% of saturation (in all aerated treatments containing live fish)
- Test termination: 12 - 19 % of saturation (the unaerated 1.52 mg/L test containers)

Salinity:

28 ‰

Nominal and measured concentrations:

- Nominal concentrations: 0 (solvent control), 0 (seawater control), 0.65, 1.1, 1.8, 3.0, and 5.0 mg/L
- Mean measured concentrations: ND, ND, 0.53, 0.93, 1.52,1.52, 2.84, 5.02 mg/L, respectively. See Table 1 in "Any other information on materials and methods incl.tables "

Details on test conditions:

TEST SYSTEM
- Test vessel: 19 L uncovered glass jars which contained 15 L of seawater
- Aeration: Yes (A duplicate set of test containers was also maintained without aeration at the middle test concentration,1.8 mg/L)
- No. of organisms per vessel: 3
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- No. of vessels per vehicle control: 4
- Biomass loading rate: 0.16 g of biomass per liter of test solution

TEST MEDIUM / WATER PARAMETERS
Water used to maintain and test the fish was natural seawater which was pumped from Big Lagoon. The pump intake was located about 80 m offshore, at a depth of approximately 3 m. Test water was pumped by a 316 stainless steel pump through hard polyvinylchloride (PVC) pipes, through sand-filled fiberglass filters, and through 10 µm pore size polypropylene core filters into an elevated fiberglass reservoir. Water flowed by gravity through PVC pipes into the laboratory. There it was pumped through a 5 µm pore size polypropylene core filter and distributed into test chambers.

WATER QUALITY MEASUREMENTS
After approximately 24, 48, 72, and 96 hours of testing, the dissolved oxygen concentration and pH were measured in each test container in which there were live animals. The salinity and temperature were measured daily in one seawater control container.

EFFECT PARAMETERS MEASURED:
- Mortality: Survival of fish was recorded at 24-hour intervals.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

2.6 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% C.L. : 2.19 - 3.29 mg/L

Details on results:

After 24 hours of exposure to the test substance, mortality of spot ranged from 0% in test concentrations ≤1.52 mg/L to 83% in the 5.02 mg/L test concentration. After 96 hours of exposure, mortality ranged from 0% in test concentrations ≤1.52 mg/L to 100% in the 5.02 mg/L test concentration.There was no significant difference between the aerated and unaerated 1.52 mg/L test concentration. The 24-hour LC50 was 3.93 mg/L with 95% confidence limits of 3.73 - 4.13 mg/L. The 96-hour LC50 was 2.60 mg/L with 95% confidence limits of 2.19 - 3.29 mg/L. The no-observed-effect concentration was 0.93 mg/L because all fish exposed to a concentration of 1.52 mg/L the test substance appeared lethargic. See Table 2 and Table 3 in "Any other information on results incl.tables " for a tabulated overwiew of the results.

Reported statistics and error estimates:

Based on the results of the test with the substance, the 24-, 48-, 72-, and 96-hour LC50's and their 95% confidence limits were calculated. The computer program estimated LC50 values by using the following three statistical methods: moving average angle analysis, probit analysis, and binomial probability (Stephan,1977). The method selected for reporting the data was determined by the characteristics of the data, that is, the presence or absence of 0% and 100% mortality and the number of concentrations in which > 0% < 100% mortality occurred.
For this test, the results of the moving average angle method were used to report the LC50 values.

Sublethal observations / clinical signs:

Table 2: Mortality of spot (Leiostomus xanthurus) exposed to the test substance in static, aerated seawater.

Mean measured concentration (mg/L)	Mortality (%)
	24 h	48 h	72 h	96 h
Control	0	0	0	0
Solvent Control	0	0	0	0
0.53	0	0	0	0
0.93	0	0	0	0
1.52	0	0	0	0
1.52 (unaerated)	0	0	0	0
2.84	8	25	33	67
5.02	83	100	100	100

 

Table 3: Calculated LC50's for spot (Leiostomus xanthurus) exposed to the test substance in static, aerated seawater, calculated based on mean measured concentrations of the test substance

Hour	LC50 (mg/L)	95% confidence limits (mg/L)
24	3.93	3.73 – 4.13
48	3.13	2.44 – 3.66
72	3.01	2.38 – 3.59
96	2.60	2.19 – 3.29

Validity criteria fulfilled:

yes

Remarks:

See the validation criteria in the "Any other information on materials and methods incl. tables"

Conclusions:

The 96-hour LC50 value was determined to be 2.60 mg/L (corresponding 95% confidence intervals of 2.19 to 3.29 mg/L).

Executive summary:

The acute toxicity of the test substance to marine spot (Leiostomus xanthurus) was studied under static conditions. The spots were maintained for 38 days in the lab conditions before the testing. The fish were exposed to the test substance dissolved in acetone (max 0.3 mL/L) at the mean measured concentrations of 0.53, 0.93, 1.52, 2.84 and 5.02 mg/L (measured by gas chromatography; the nominal concentrations were 0.65, 1.1, 1.8, 3.0 and 5.0 mg/L, respectively).The fish (12 fish per concentration) were incubated at a temperature of 22 - 23°C, 7.4 - 8.1 pH, salinity 27 - 28‰ and dissolved oxygen ≥ 74% of saturation. A duplicate test vessel at a concentration of 1.52 mg/L was maintained without aeration. There were also blank and vehicle control in the test. Mortality, general symptoms of toxicity, dissolved oxygen concentration and pH were observed 0, 24, 48, 72 and 96 hours after dosing. Temperature and salinity were daily measured in one seawater control container.

One fish died in the vehicle control but in the blank control there was no mortality during the study. After 96 hours the mortality varied between 0% at the three lowest concentrations to 100% at the highest concentration. There was no toxic symptoms in the controls and at the concentrations of 0.53 and 0.93 mg/L but at the concentrations over 1.52 mg/L all the fish were lethargic. After 96 hours the LC0-, LC50-, and LC100- value was 1.52, 2.6 (95% confidence limit 2.19 - 3.29 mg/L) and 5.02 mg/L, respectively, based on the mean measured concentrations.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Jun 1999 to 7 Jul 1999

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:

yes

Remarks:

Fish length: mean 62 mm (range 53-65 mm)

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Qualifier:

according to guideline

Guideline:

EPA OPP 72-1 (Fish Acute Toxicity Test)

Deviations:

yes

Remarks:

Number of replicates, of test organisms, continuous monitoring of temperature and equilibration time

GLP compliance:

yes

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

4.3 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Sublethal observations / clinical signs:

The measured concentrations varied 100 - 109% of the nominal ones at the start of the test and 89 - 104% at the end of the study. After 96 hours exposure 100% mortality was observed at the concentrations of 5.8 and 10 mg/L. The LC50 (96 h) was determined to be 4.3 mg/L based on the nominal concentrations.The mortality in the blank and vehicle controls was 0 %. NOEC value was 1.0 mg/L. On the basis of the results the test substance is acutely toxic to fish.

Validity criteria fulfilled:

yes

Conclusions:

The LC50 (96h) of the test substance was determined to be 4.3 mg/L based on nominal concentrations. Using the classification range given in Annex VI of Directive 67/548/EEC, the test substance is "toxic to Rainbow Trout" based on the inherent toxicity of the test substance in this test. The highest concentration with no sublethal and lethal effects was 1.0 mg/L. Based on the similarity of the measured concentrations of the test substance at the start and the end of the exposure, the test item was stable in the test solutions over the period of the test under static conditions.

Executive summary:

The acute toxicity test was performed to assess the effect of the test substance on the mortality of the rainbow trout (Oncorhynchus mykiss) under static conditions following OECD Guideline No. 203, 92/69/EEC C.1 and EPA FIFRA Guideline NO. 72 -1 in compliance with GLP criteria. Nominal concentrations were 1.0, 1.8, 3.2, 5.8 and 10 mg/L.

After the preparation of the test concentrations and an equilibration time of 4.5 hours, 7 fish were randomly placed in each of the 20 L glass aquaria containing 15 L of test solution (beginning of exposure). Fish were exposed to the test substance for 96 hours and were observed for adverse effects at 2, 24, 48, 72 and 96 hours. The temperature was maintained constant at 13.3-13.4°C and the dissolved oxygen content between 81-103 % of the saturation value by gentle aeration during exposure. The pH was determined to be 7.9 - 8.2 and the total hardness 178 mg CaCO3/L.

The test item appeared homogeneously distributed in the test vessels at all test concentrations over the period of the test. No precipitation was observed in the stock solutions or the testing solutions throughout the duration of the test.
The nominal concentrations were 1.0, 1.8, 3.2, 5.8, 10 mg test item/L. Measured test concentrations of the test substance at the beginning and the end of the exposure period ranged from 100 - 109% and 89 - 104% of the nominal concentrations, respectively. At 5.5 and 10 mg test item/L, the measured test concentrations at the end of the exposure were determined after 24 h when 100 % mortality occurred. After 96 h exposure,100% mortality occurred at concentrations of 5.8 and 10 mg/L. The LC50-value were observed to be 4.3 mg/L during the period 24 to 96 h exposure. The LC0 (96h) was 3.2 mg/L and the LC100 (96h) 5.8 mg/L. NOEC value was 1.0 mg/L. On the basis of the results the test substance is acutely toxic to fish.

Description of key information

All available data was assessed. For toxicity to freshwater fish, the reliable effect value representing the worst-case effects was included here and used in the CSA. For toxicity to marine water fish, the study used standard test conditions was selected as the key study. Other studies are included as supporting information. 

Freshwater; 96-h LC50 = 4.3 mg/L; Oncorhynchus mykiss; OECD TG 203; Rufli, 1999

Marine water; 96-h LC50 = 2.60 mg/L (corresponding 95% confidence intervals of 2.19 to 3.29 mg/L); Leiostomus xanthurus; EG; Ward, 1982

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

4.3 mg/L

Marine water fish

Marine water fish

Dose descriptor:

LC50

Effect concentration:

2.6 mg/L

Additional information

 There are nine studies on freshwater fish and two studies on marine water fish available for this endpoint. For toxicity to freshwater fish, the reliable effect value representing the worst-case effects was included here and used in the CSA. For toxicity to marine water fish, the study used standard test conditions was selected as the key study.

For toxicity to freshwater fish, the 96-h study on Oncorhynchus mykiss (Rufli 1999, OECD TG 203, GLP, Reliability 1) was selected as the key study since the effect value representing the worst-case effects (i.e. showed the lowest LC 50 value). For toxicity to marine water fish, the 96-h study on Leiostomus xanthurus (Ward 1982, no standard guideline followed, not GLP, Reliability 2) was selected as the key study since it used used standard test conditions. 

In the freshwater key study, seven fish per group (Oncorhynchus mykiss, no replicates) were exposed to nominal concentrations of 0 (control), 0 (vehicle, DMF), 1.0, 1.8, 32., 5.8 and 10 mg/L (mean measured concentrations: 1.0, 1.7, 3.1, 5.8 and 10.7 mg/L, respectively) in a static system. The exposure conditions were: temperature 13.3 - 13.4 °C, pH 7.9 - 8.2, oxygen saturation of 81 - 103% and 16 hours daily light. The 96h-LC50 was calculated to be 4.3 mg/L based on nominal concentrations. LC50 of 0.85 mg/L for Oncorhynchus mykiss was found in the study from Thompson et al.(1980b). The test concentrations were measured only at the beginning of the test, and the effect value was based on nominal concentrations. Thus, the study is used as supporting information. The other studies on freshwater fish (Lepomis macrochirus, Ictalurus punctatus, Pimephales promelas, Oncorhynchus mykiss, Cyprinus carpio and Ictalurus melas) support the results with 96-h LC50 ranging from 1.3 to 6.8 mg/L based on nominal or mean measured concentrations (Thompson 1980 a and c, Shaw 2018, Hotz 1982a and b and De morsier 1982a and b, Reliability 1 or 2).

In the marine water key study, twelve fish per group (Leiostomus xanthurus, 4 replicates, 3 fish per replicate) were exposed to nominal concentrations of 0 (blank control), 0 (solvent control: acetone), 0.65, 1.1, 1.8, 3.0 and 5.0 mg/L (mean measured concentration: ND, ND, 0.53, 0.93, 1.52, 1.52, 2.84 and 5.02 mg/L, respectively) in a static system. The exposure conditions were as follows: temperature 22 - 23°C, pH 7.4 - 8.1, salinity 27 - 28‰ and dissolved oxygen ≥ 74% of saturation. The 96hLC50 was calculated to be 2.6 mg/L, based on mean measured concentrations. The second study on marine water fish (Leiostomus xanthurus) showed a lower effect value with 96h LC50 of 2.2 mg/L based on measured initial concentrations. The water was not aerated in this study and the dissolved oxygen level was low (≥ 14% of saturation), which may have an effect on the test result. Thus, this study was included as a supporting study (Ward 1981, Reliability 2).