3,5-dimethylpyrazole

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 July 2012 to 25 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentrations and the control were sampled for analysis.
- Sampling method: Singular 2 mL samples were collected at 0 and 48 hours from the approximate centre of each test vessel. At the end of the exposure period, the replicates were pooled at each concentration before sampling. Additional reserve samples were stored frozen for possible future analysis.
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with the highest concentration of 100 mg/l applying 35 minutes of magnetic stirring to ensure complete dissolving of the test substance in the test medium. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium.
- Controls: A blank control of untreated test medium was employed.
- Evidence of undissolved material: The final test solutions were all clear and colourless.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: In-house laboratory culture with a known history.
- Health: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Age at study initiation (mean and range, SD): Young daphnids, < 24 hours, from parental daphnids of more than two weeks old.
- Weight at study initiation (mean and range, SD):
- Length at study initiation (length definition, mean, range and SD):
- Method of breeding: Breeding batches were started with approximately 250 newborn daphnids, i.e. less than 3 days old, were placed in 5 litres of M7 medium at 18 – 22ºC. The maximum age of the cultures was 4 weeks. Daphnids were feed a suspension of fresh water algae daily. Half of the culture medium was replaced every 7 days.
- Feeding during test: No feeding during exposure.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

none

Hardness:

180 mg/L as CaCO3

Test temperature:

The temperature of the test medium was 20.2 ºC at the begning of the test, the temperature then varied from 19.2 ºC to 19.8 ºC during exposure.

pH:

The pH measured 8.1 at the beginning of exposure and 8.2 at the end in both the control and 100 mg/L test group.

Dissolved oxygen:

Dissolve oxygen in the control and 100 mg/L test group measured 8.8 mg/L at the beginning and 8.8 and 8.7 mg/L end of exposure, respectively.

Salinity:

-

Nominal and measured concentrations:

Nominal concentration: 100 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL all glass vessels.
- Volume of test solution per vessel: 80 mL
- Aeration: No aeration of the test solution.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 replicates at 100 mg/L.
- No. of vessels per vehicle control: 4 replicates.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO M7 medium. Analytical grade chemicals were dissolved in tap water purified by reverse osmosis, see Table 1 for a detailed list of composition.

OTHER TEST CONDITIONS
- Photoperiod: A 16 hour light photoperiod was maintained.

EFFECT PARAMETERS MEASURED
- Immobility and mortality: Recorded at 24 and 48 hours.
- pH and dissolved oxygen: Measured in the 100 mg/L (highest concentration) and control at the beginning and end of exposure.
- Temperature of medium: Measured continuously in the temperature control vessel.

TEST CONCENTRATIONS
- The study was performed as a limit test combined with a range-finding test.
- Range finding study
- Test concentrations: Nominal concentrations of 0.10, 1.0 and 10 mg/L.
- Replications: All concentrations were tested in duplicate.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: concentration analytically confirmed

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: concentration analytically confirmed

Details on results:

- Concentration analysis:
The concentration of the test material medium was confirmed to be stable and in agreement with the nominal concentration by 103 to 104%. The mean recovery of the procedural recovery samples at the concentration of 100 mg/L fell within the criterion of 70-110%. The mean recovery of the procedural recovery samples at the concentration of 0.1 mg/L was slightly high at 115%. Since the deviation above 110% was small, it was accepted. Results are presented in Table 3.

Results with reference substance (positive control):

Daphnids were exposed the reference substance for 48 hours at 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L, a blank control was also included.
The responses were in the lower range of the expected historical data.
EC50 = 0.30 mg/L with a 95% confidence interval between 0.28 and 0.35 mg/L.

Reported statistics and error estimates:

Calculations:
No EC50 could be calculated as the test material proved to be non-toxic i.e. EC50 > maximum concentration tested.

Table 2. Acute Immobilisation of Daphnids During Exposure

Concentration (mg/L)	Vessel Replicate	Number of Exposed Daphnids	Response at 24 hrs		Response at 48 hrs
			Number	Total %	Number	Total %
Control	A	5	0	0	0	0
	B	5	0		0
	C	5	0		0
	D	5	0		0
0.10	A	5	0	0	0	0
	B	5	0		0
1.0	A	5	0	0	0	0
	B	5	0		0
10	A	5	0	0	0	0
	B	5	0		0
100	A	5	0	0	0	0
	B	5	0		0
	C	5	0		0
	D	5	0		0

Table 3. Concentration Analysis of the Test Material in the Test Medium

Time of sampling [hours]	Concentration		Relative to nominal [%]	Relative to initial [%]
	Nominal [mg/l]	Analysed [mg/l]
0	0	n.d.	n.a.
	100	103	103
48	0	n.d.	n.a.	101
	100	104	104

¹       Samples were stored in the freezer (≤ -15°C) until the day of analysis.

n.d.  Not detected.

n.a.   Not applicable.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, the test material did not induce acute immobilisation in Daphnia magna up to 100 mg/L. Therefore the NOEC was determined to be 100 mg/L and the EC50 can be said to exceed the maximum concentration tested, > 100 mg/L.

Executive summary:

The short term toxicity to aquatic invertebrates was determined in a 48 hour static freshwater toxicity study performed using Daphnia magna. The study was performed in line with GLP and according to the standardised guidelines OECD 202 and EU Method C.2.

The test organisms were exposed to the test material at the following nominal concentrations; 0.10, 1.0, 10 and 100 mg/L, with a concurrent untreated control. Observed for immobility and mortality were made at 24 and 48 hours after the start of exposure. The 100 mg/L concentration was confirmed to be stable and in agreement with the nominal concentration by 103 to 104%. Potassium dichromate was run as a positive control, results of which were in agreement with historical data, confirming the sensitivity of the batch of Daphnia magna used.

Under the conditions of the test, the test material did not induce acute immobilisation in Daphnia magna up to 100 mg/L. Therefore the NOEC was determined to be 100 mg/L and the EC₅₀ can be said to exceed the maximum concentration tested, > 100 mg/L.

Description of key information

The toxicity of 3,5-Dimethylpyrazole to aquatic invertebrates has been determined to be EC50 > 100 mg/L and NOEC 100 mg/L in a key study (Migchielsen, 2012a) performed according to OECD 202 and EU Method C.2.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

Migchielsen (2012a) has been provided as the key study where the acute toxicity of the test material to aquatic invertebrates was determined to be EC₅₀> 100 mg/L with a NOEC of 100 mg/L. The study was performed to GLP and standardised guidelines and had thus been assigned a reliability score of 1 using the principles for assessing data quality as set out in Klimisch (1997).