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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Jun 1999 to 28 Jun 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

1984

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Qualifier:

according to guideline

Guideline:

EPA OPP 72-2 (Aquatic Invertebrate Acute Toxicity Test)

Version / remarks:

1989

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Water samples of each concentration (2 x 50 mL volume each) were taken from the freshly prepared test solutions at the start of exposure. At test termination (48 h) samples of 50 mL were taken from 3 replicates of the treatment group and analysed for CGA 64250. All samples were kept at -18°C to -
22°C until analysis.

Vehicle:

yes

Remarks:

DMF

Details on test solutions:

3.201 g test item were mixed and made up to 10 mL with DMF 5 mL of this stock solution were mixed and made up to 10 mL with DMF to prepare stock solution 2.
5 mL of stock solution 2 were mixed and made up to 10 mL with DMF to prepare stock solution 3.
5 mL of stock solution 3 were mixed and made up to 10 mL with DMF to prepare stock solution 4.
5 mL of stock solution 4 were mixed and made up to 10 mL with DMF to prepare stock solution 5.
5 mL of stock solution 5 were mixed and made up to 10 mL with DMF to prepare stock solution 6.
50 μL of each stock solution were mixed with 1 L M4-Medium to prepare the test solutions. In addition calculated amounts of vehicle to achieve identical vehicle concentrations at all test concentrations were added.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water fleas
- Strain: Clone 5
- Age at study initiation: < 24 hours
- Culture: Cultures of Daphnids were maintained in glas vessels of 3 L containing approximately 2.8 L of M4 medium. About two third of this culture medium was renewed three times a week. The daphnids were fed daily with the exception of the weekend with a suspension of green algae (1.7 mL/L of a Scenedesmus subspicatus suspension with a density of approximately 0.2E+08 cells/mL) supplemented by a suspension of TETRAMIN-extract in such quantities that the food is consumed after 24hours. Friday the amount of green algae was doubled. Culturing was performed in a constant environment room at 20 ± 2°C and a photo period of 16 hours light and 8 hours dark, with a transition period of 30 minutes. Three times a week the young daphnids were separated from the original culture to maintain a homogeneous stock of animals within the same age.
- Feeding during test: No feeding was perfonned during the 48 h test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

261 mg/L as CaCO3

Test temperature:

21 ± 1 °C

pH:

pH 8.2 - 8.4

Dissolved oxygen:

- At the beginning of the test: 98 - 101% of saturation
- At the end of the test: 82 - 84% of saturation

Conductivity:

649 μS/cm

Nominal and measured concentrations:

- Nominal concentrations: 0.5, 1.0, 2.0, 4.0, 8.0 and 16 mg/L
- Measured concentrations at the start: 0.5, 1.0, 1.9, 3.8, 8.2 and 16.3 mg/L, respectively
- Measured concentrations at the end: 0.5, 1.0, 2.0, 3.7, 8.4 and 16.3 mg/L, respectively

Details on test conditions:

TEST SYSTEM
- Test vessel: 150 mL glass beakers
- Type: Open
- Fill volume: 100 mL
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- No. of vessels per vehicle contro: 4

TEST MEDIUM / WATER PARAMETERS
- Test medium: M4-medium according to ELENDT
- Measurement of water parameters: The oxygen content, pH and temperature were measured at test start and at test end (48 h). Hardness and conductivity of the test medium were determined from the freshly prepared M4-medium.

OTHER TEST CONDITIONS
- Light intensity: Light-dark cycle of 16h light : 8h dark with a 30 min. transition period (1200 lux)

EFFECT PARAMETERS MEASURED
Immobilization and other behavioural c hanges of the daphnids were recorded,after 24 and 48 hours
of exposure. Those animals not able to swim within IS seconds after gentle agitation of the test
container were considered to be immobile.

Reference substance (positive control):

no

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

10.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other:

Remarks:

Immobilization and behavioural changes

Remarks on result:

other:

Remarks:

95% C.L.: 8.6 - 12.2 mg/L

Details on results:

An overview of the results is provided in Table 1 in 'Any other information on results incl. tables'
The test concentrations were 95 - 103% of the nominal ones at the start of the exposure and 93 - 105% at the end. There were no mortalities observed in the control. After 24 and 48 hours of exposure, no immobilization of daphnids and no sublethal effects were observed at the concentrations of 0.5, 1.0, 2.0 and 4.0 mg/L. At the test concentrations of 8.0 and 16.0 mg/L, 15% and 40% of daphnids were immobilized after 24 hours and 25% and 90% after 48 hours of exposure. The EC50 (48 h) -value was determined to be 10.2 mg/L (95% confidence limit 8.6 - 12.2 mg/L) based at the nominal concentrations and NOEC-value 4.0 mg/L. On the basis of the results the test substance is acutely moderately toxic to Daphnia.

Table 1. Immobilization of daphnia after 48 hours of exposure

 

	Replicate				total	%
Nominal conc. mg/L	1	2	3	4
Blank	0	0	0	0	0	0
Vehicle	0	0	0	0	0	0
0.50	0	0	0	0	0	0
1.0	0	0	0	0	0	0
2.0	0	0	0	0	0	0
4.0	0	0	0	0	0	0
8.0	0	2	1	2	5	25
16	5	4	5	4	18	90

Validity criteria fulfilled:

yes

Remarks:

< 10% of the daphnids in the controls should be immobilised at the end of the exposure.

Conclusions:

Based on the findings, the 48-h EC50 to the test sunsbance is 10.2 mg/L (95 % confidence limit 8.6 - 12.2 mg/L).

Executive summary:

The acute toxicity study of the test substance on Daphnia magna was performed in accordance with OECD Guideline No.202 (1984), EPA Guideline 850.1010 and EPA FIFRA Guideline No. 72-2 (1989) and it was in compliance with GLP. Test concentration of nominal 0.5, 1.0, 2.0, 4.0, 8.0 and 16 mg/L of the test substance were selected based on ecotox results published in “The Pesticide Manual", 1998 (EC50 Daphnia 4.8 mg/L). Exposure started when the daphnids were placed in the test vessels containing the test solutions. Twenty daphnids were exposed to each of the desired test concentrations under static conditions in M4 - medium. For the test concentrations and the controls (blanc and vehicle), four replicates were performed. Each replicate consisted in five specimens in 100 mL test solution. The daphnids were observed for immobilization and behavioural changes after 24 h and 48 h of exposure. The water temperature was maintained constant throughout the duration of the test at 21 ± 1°C in all test concentrations. The oxygen saturation was measured to be 98 - 101% and 82 - 84% at the start and at the end of exposure, respectively. The pH was 8.4 - 8.5 and 8.2 at the start and at the end of exposure, respectively.
The test item was homogeneously distributed in the test vessels at all test times and test concentrations. The measured test concentrations of the test substance were 0.5, 1.0, 1.9, 3.8, 8.2 and 16.3 mg/L and 0.5, 1.0, 2.0, 3.7, 8.4 and 16.3 mg/L at the start and the end of exposure, respectively, i.e. 95 - 103 % of the nominal concentrations at the start and within 93 - 105 % at the end of exposure, respectively.
After 24 and 48 hours of exposure, no immobilization of daphnids and no sublethal effects were observed at test concentrations of 0.5, 1.0, 2.0 and 4.0 mg/L. At test concentrations of 8.0 and 16 mg/L 15% and 40% of daphnids were immobilized after 24 hours and 25% and 90% after 48 hours of exposure. Based on the findings, theEC50 (48 h) of the test substance was determined to be 10.2 mg/L (95% confidence limit 8.6 - 12.2 mg/L). The highest concentration with NOEC after 48 hours was 4.0 mg/L.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Jul 1981 to 27 Jul 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

The test was performed before GLP requirements

Qualifier:

no guideline followed

Principles of method if other than guideline:

Method was performed in accordance with the test facility's test protocol for Mysid Shrimp "Actue and Life-Cycle Toxicity Tests with Mysid Shrimp (Mysidopsis bahia) ", dated May 1981.

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

Samples for chemical analyses of the test substance in seawater were taken from alternate duplicates of each treatment on days 0 and 4.
A 100 mL water sample was collected through a fixed glass siphon which sampled from near the center of the aquarium and at mid-depth in the water column. The samples were collected into amber glass bottles with Teflon-lined screw caps.

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

The chemical stock solution (11.4 g of active ingredients of the test substance per 100 mL of reagent grade acetone) was delivered into a mixing chamber from a 50 mL glass syringe and from there it siphoned down into the chemical cells. A Teflon solenoid valve, controlled by two float switches, regulated seawater flow into the water cells. Mixing of various volumes of uncontaminated dilution seawater from the water cells with various volumes of test solution in the chemical cells produced the five concentrations of of the test substance which were distributed to the appropriate test containers.

Test organisms (species):

Americamysis bahia (previous name: Mysidopsis bahia)

Details on test organisms:

TEST ORGANISM
- Common name: Mysid shrimp
- Source: The test animal were cultured at the test facility
- Acclimation condition: Juvenile mysids were isolated from adults and were acclimated in flowing, natural seawater until testing was initiated
- Age at study initiation: 6 - 8 days old (estimated) and appeared to be in excellent condition
- Feeding and food type during test: 48-hour old Artemia nauplii once daily.

Test type:

flow-through

Water media type:

saltwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

26 °C

pH:

Test initial and termination: 7.8 (in all treatments)

Dissolved oxygen:

- During experimental period: 4.8 - 5.9 mg/L
- Test initial : ≥ 84% of saturation
- Test termination: ≥ 72% of saturation

Salinity:

28 - 31 ‰

Nominal and measured concentrations:

- Nominal concentrations: 0 (solvent control), 188, 375, 750, 1500 and 3000 µg/L
- Mean measured concentrations: ND, 158, 456, 753, 1402, 2900 µg/L, respectively. See Table 1 in "Any other information on materials and methods incl.tables "

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass Petri dishes to which a 15 cm high nylon screen collar (315 µm mesh size opening) was attached with silicone sealant
- Test aquarium: Two test vessels were placed in each of two sections of one aquarium of all concentrations and the control (two true duplicates)
- Volume of solution: Approximately 9 L/aquarium
- Type of flow-through: An intermittent-flow system by using a proportional diluter (Mount and Brungs, 1967). Dilution delivery was 0.5 L/cycle/test aquarium at a dilution rate of 50%. The average number of cycles was approximately 6 per hour.
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per vehicle control : 4
- No. of vessels per seawater control: 0

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Test water was natural seawater which was pumped from Big Lagoon, a Gulf of Mexico estuary adjacent to the test facility. The pump intake was about 80 m offshore at a depth of approximately 3 m. Seawater 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 was aerated in the reservoir and flowed by gravity through PVC pipes into the laboratory.
- Intervals of water quality measurement: Salinity, temperature, pH and dissolved oxygen were measured daily.

EFFECT PARAMETERS MEASURED
- Signs of toxicity: In order to observe mysids, each vessel was gently lifted from the aquaria and then allowed to drain through the screen to the depth of the Petri dish. The vessel was then placed on a back-lighted table.
- Mortality: During the test, the number of dead animals were recorded at 24-hour intervals.

VEHICLE CONTROL PERFORMED: Yes

RANGE-FINDING STUDY: Selection of concentrations for the test were based on the results of a 48-hour static test.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

510 µg/L

95% CI:

>= 370 - <= 670

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Details on results:

On day 0 of the flow-through test, the measured concentrations of the test substance ranged from 98% to 155% of the nominal concentrations. Chemical measurements on day 4 of the test demonstrated the test concentrations were between 70% and 89% of the nominal concentrations.
After 24 hours of exposure to the test substance in the flow-through system, mortality of mysid shrimp ranged from 0% in test concentrations ≤ 753 µg/L to 90% in the 2,900 µg/L test concentration. After 96 hours of exposure, mortality ranged from 10% in the 158 µg/L test concentration to 100% in the 2,900 µg/L test concentration. There was 5% mortality in the solvent control. See Table 2 in "Any other information on results incl.tables" for a tabulated overview of the results.
The calculated 24 hour LC50, based on measured test concentrations, was 1,700 µg/L with 95% confidence limits of 1,500 - 2,100 µg/L. The 96-hour LC50 was 510 µg/L with 95% confidence limits of 370 - 670 µg/L based on measured test concentrations. See Table 3 in "Any other information on results incl.tables " .

Reported statistics and error estimates:

The computer program estimated LC50 values by using one of three statistical methods in the following order: moving average angle analysis, probit analysis, or binomial probability. The method selected 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 (Stephan, 1977). The computer scanned the data, identified the most suited method, and performed the analyses. The moving average angle method was used to calculate all LC50 values reported here.

Table 2: Mortality ofmysid shrimp (Mysidoysis bahia) exposed to the test substance for 96 hours in flowing seawater. Salinity was 30±1‰ and temperature was 26°C.

Average measured concentration (mg/L)	Mortality (%)
	24 h	48 h	72 h	96 h
Solvent Control	0	0	5	5
0.158	0	0	10	10
0.456	0	5	20	55
0.753	0	15	45	55
1.402	30	85	90	95
2.900	90	100	100	100

 

Table 3: Calculated LC50's for mysid shrimp (Mysidopsis bahia) exposed to the test substance for 96 hours in flowing seawater. Salinity was 30±1‰ and temperature was 26°C. Values were calculated based on average measured concentrations of test material in seawater.

Hour	LC50 (mg/L)	95% confidence limits (mg/L)
24	1.700	1.500 – 2.100
48	1.000	0.860 – 1.200
72	0.670	0.490 – 0.920
96	0.510	0.370 – 0.670

 

Validity criteria fulfilled:

yes

Remarks:

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

Conclusions:

The 96-hour LC50 value was determined to be 0.510 mg/L (corresponding 95% confidence limits of 0.370 to 0.670 mg/L).

Executive summary:

The acute toxicity of the test substance to 6 - 8 days old mysid shrimp (Mysidopsis bahia) was studied in a 96-hour intermittent flow-through test. The shrimps were acclimatised before the test in natural seawater and thereafter they (20 shrimps per dose level and 5 per petri dish) were exposed to the acetone dissolved compound at the mean measured concentrations of 0.158, 0.456, 0.753, 1.402, and 2.900 mg/L (measured by gas chromatography; the nominal concentrations were 0.188, 0.375, 0.750, 1.500, 3.000 mg/L, respectively). Samples for chemical analyses of the test substance were taken from alternate duplicates of each treatment at the beginning and at the end of the study. The properties of the test water were: temperature 26 °C, pH 7.8 - 7.9, dissolved oxygen concentration 4.8 - 5.9 mg/L and salinity 28 - 31‰. The water parameters were measured daily. The mortality of the shrimps was recorded after 24, 48, 72 and 96 hours exposure.

There was 5% mortality in the solvent control. After 96 hours of exposure, mortality ranged from 10% in the 0.158 mg/L test concentration to 100% in the 2.900 mg/L test concentration. Based on these findings, the LC0 -, LC50 -, and LC100 - values were < 0.158, 0.510 (95% confidence limits 0.370 – 0.670 mg/L) and 2.900 mg/L, respectively. All lethal concentrations were calculated based on the mean measured concentrations.

Description of key information

All available data was assessed and the reliable effect values representing the worst-case effects are included here and used in the CSA. Other studies are included as supporting information.

 Freshwater, 48-h EC50 = 10.2 mg/L (based on mean measured concentrations); Daphnia magna; Immobilization and behavioural changes; OECD TG 202; Grade, 1999 

 Marine water; 96-h LC50 = 0.510 mg/L (based on mean measured concentrations); Mysidopsis bahia; No guideline followed; Hollister, 1981  

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

LC50

Effect concentration:

10.2 mg/L

Marine water invertebrates

Marine water invertebrates

Dose descriptor:

EC50

Effect concentration:

0.51 mg/L

Additional information

 There are five studies available for this endpoint. The 48-h study on daphnia (Grade 1999, OECD TG 202, GLP Reliability 1) and 96-h study on mysid shrimp (Hollister 1981, no standard guidelines followed, not GLP, Reliability 2) were selected as key studies. The effect values in these two studies are reliable and represent the worst-case effects (i.e. showed the lowest E(L)C50 value) for freshwater and marine water conditions. The other studies are included as supporting information.

In the freshwater key study, daphnia (5 daphnias per replicate, 4 replicates/concentration) were exposed to the test substance at nominal concentrations of 0.5, 1.0, 2.0, 4.0, 8.0 and 16 mg/L in a static system for 48 hours. The measured test concentrations of the test substance were 0.5, 1.0, 1.9, 3.8, 8.2 and 16.3 mg/L and 0.5, 1.0, 2.0, 3.7, 8.4 and 16.3 mg/L at the start and the end of exposure, respectively. A blank control and a solvent control (dimethylformamide) were included. The exposure conditions were: temperature 21 °C, pH 8.4 - 8.5, dissolved oxygen 82.3 – 100.3% of saturation. The 48-h EC50 was estimated to be 10.2 mg/L, based on mean measured concentrations. A Lower effect value (LC50 = 4.8 mg/L based on nominal concentrations) was found in a study by Forbis et al. (1980). However, considering the measured concentrations ranged from 43% to 153% in this study, the study is used as supporting information. Therefore, the EC50 of 10.2 mg/L is used in the CSA for this endpoint. The other freshwater study supports the result with 96 h LC 50 of 42 mg/L for catfish (LeBlance & Mastone 1981, EPA 660/3-75-009, GLP, Reliability 2).  

In the marine water key study, 6 - 8 days old mysid shrimp (Mysidopsis bahia) were exposed to the compound (20 shrimps per dose level and 5 per petri dish) at mean measured concentrations of 0.158, 0.456, 0.753, 1.402, and 2.900 mg/L ( the nominal concentrations were 0.188, 0.375, 0.750, 1.500, 3.000 mg/L, respectively) in a flow-through system. Test conditions were as follows: temperature 26 °C, pH 7.8 - 7.9, dissolved oxygen concentration 4.8 - 5.9 mg/L and salinity 28 - 31‰. The 96-hour LC50 value was determined to be 0.510 mg/L based on mean measured concentrations. In the other marine water study, the 96 h EC50 for oysters was determined to be 1.7 mg/L (Ward 1981, no guideline followed, not GLP, Reliability 2).