3-methylpyrazole

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06.04.-16.08.2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Molecular mass (g mol-1) 82.1
Date of receipt / amount: 05 February 2021 / 50 mL
Expiry date: March 2023
Analysed purity: 98.8%
Density: 1.0203 g/cm3
Appearance: yellow-orange liquid
Solubility: soluble

Analytical monitoring:

yes

Details on sampling:

Sampling for analysis was performed at test start, once a week (week 1, week 2 and week 3 after test start) at renewal processes and at test end for the fresh and spent test solutions.

Vehicle:

yes

Details on test solutions:

The test item 3-Methylpyrazole was soluble in the test medium. An aqueous stock solution was prepared by adding the test item to test medium, followed by stirring for 1 minute on a magnetic stirrer. The concentrations were prepared by serial dilutions of the stock solution A.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test organism: Daphnia magna STRAUS
Origin of the test organism: Landesanstalt für Umweltschutz Baden-Württemberg,
Griesbachstr. 1, 76185 Karlsruhe, Germany
Origin of the animals used in the test: holding and breeding occurred in the Test Facility under standardised laboratory conditions; test animals were derived from a healthy stock; no first brood progeny was used as test animals
Age of the Daphnia (at test start): < 24 hours and not first brood progeny
Breeding conditions: breeding was performed in glass beakers (50 mL) under the same conditions as in the test. Test animals were separated from the adults by means of a pipette.
Breeding medium: Elendt M4 medium
(according to the OECD Guideline 211, Annex 2)
Food: Alga Desmodesmus subspicatus purchased from MBM ScienceBridge GmbH, Hans-Adolf-Krebs-Weg 1, 37077 Göttingen, Germany,
Feeding: daily, depending on age the Daphnia were fed with a defined suspension of algae:
≤ 1 day old: 0.5 mL/50 mL M 4
3 to 10 days old: 1.0 mL/50 mL M 4
> 10 days old: 1.5 mL/50 mL M 4

Test type:

semi-static

Water media type:

other: Elendt M4 medium (according to the OECD Guideline 211, Annex 2) Prepared at least one day in advance

Limit test:

no

Total exposure duration:

21 d

Hardness:

180 - 182 mg/L CaCO2

Test temperature:

20.0 – 20.5 °C (continuously measured)

pH:

no adjustment of pH
7.035-7.841

Dissolved oxygen:

7.67-9.32 mg/L

Conductivity:

conductivity ≤ 10 µS/cm, measured: 1.0 µS/cm

Nominal and measured concentrations:

Nominal: 1.23, 3.71, 11.1, 33.3 and 100.0 mg/L
The measured test concentrations of 3-Methylpyrazole in test solutions ranged from 97 to 106% in fresh samples and from 98 to 109% in aged samples respectively with one exception.
One recovery was not in the range of 80 to 120% of nominal because of a technical error (155% in the spent test solution at the nominal test item concentration of 1.23 mg/L test item at day 7 after test start). Therefore, the retain samples was analysed and a recovery of 125% of nominal was found. This value was determined as outlier value.
Based on the above, the results presented are based on the nominal concentrations for the test item.

Details on test conditions:

Test conditions
Test vessel: glass beakers (approximately 60 mL)
Number of parent (Daphnia)/
test vessel (= replicate): 1
Number of test vessels/
concentration: 10 + 3 additional vessels without animals (1 for oxygen and pH measurements, 1 for analysis and 1 for retained specimens)
Number of daphnids/concentration: 10
Loading: 50 mL test solution per daphnid
Test medium: Elendt M4 medium
(according to the OECD Guideline 211, Annex 2)
Prepared at least one day in advance
Preparation: with air-saturated deionized water
(conductivity ≤ 10 µS/cm, measured: 1.0 µS/cm)
Test procedure: semi-static test
no aeration of the vessels
no adjustment of pH
feeding during the test period
Feeding: three times per week (medium renewals) with living algal cells of Desmodesmus subspicatus; the supplied diet (concentrated algal suspension) was based on the amount of organic carbon (C) provided to each parent animal: between 0.1 and 0.2 mg C/Daphnia/day
Climatic conditions
Temperature: 20.0 – 20.5 °C (continuously measured)
Light-dark-cycle: 16 h light : 8 h dark, daily
light intensity: approximately 20 μE/m2s-1,
equivalent to 1000 lux
Test duration: 21 days

Reference substance (positive control):

not required

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

3.71 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality: Total number living offspring produced at the end of the test per parent daphnia at the start of the test excluding from the analysis parental accidental and/or inadvertent mortality {TG 211}

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

33.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

19 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality: Total number living offspring produced at the end of the test per parent daphnia at the start of the test excluding from the analysis parental accidental and/or inadvertent mortality {TG 211}

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

19.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

57.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 33.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 33.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

Mean cumulative number of offspring per introduced parent
The LOEC (lowest observed effect concentration) for mean cumulative number of offspring per introduced parent at 21 days after application was determined to be 11.1 mg/L test item. Hence, the NOEC (no observed effect concentration) was determined to be 3.71 mg/L test item, nominal. An EC10 value of 5.00 mg/L test item, an EC20 of 7.91 mg/L test item and an EC50 of 19.0 mg/L test item based on nominal concentrations, were calculated.
Mean cumulative number of offspring per survived parent
The LOEC (lowest observed effect concentration) for mean cumulative number of offspring per survived parent at 21 days after application was determined to be 3.71 mg/L test item. Hence, the NOEC (no observed effect concentration) was determined to be 1.23 mg/L test item, nominal. An EC10 value of 4.29 mg/L test item, an EC20 of 7.20 mg/L test item and an EC50 of 19.3 mg/L test item based on nominal concentrations, were calculated.
Mortality of parents
The control mortality of parent daphnids was 0%. Parental mortality occurred at the highest tested concentration of 100.0 mg/L test item, nominal at day 9-14 after test start (100%). The LOEC for mortality of parent animals was determined to be 100.0 mg/L test item, nominal. Hence, a NOEC of 33.3 mg/L test item, nominal was determined.
An EC10 value of 51.0 mg/L test item, nominal, an EC20 of 53.2 mg/L test item and an EC50 of 57.5 mg/L test item, based on nominal concentrations were calculated.


Age of first reproduction
The LOEC for the age of first reproduction was determined to be higher than 33.3 mg/L test item, nominal Hence, a NOEC of higher or equal 33.3 mg/L test item based on nominal concentrations was determined. EC10, EC20 and EC50 values were higher than 33.3 mg/L test item.
Development rate for reproduction
The LOEC for the development rate for reproduction was determined to be higher than 33.3 mg/L test item, nominal Hence, a NOEC of higher or equal 33.3 mg/L test item based on nominal concentrations was determined. EC10 EC20 and EC50 values of higher than 33.3 mg/L test item, based on nominal concentrations were calculated.
Intrinsic rate (r) of population increase
The LOEC for intrinsic rate of population increase was determined to be 11.1 mg/L test item. Hence, a NOEC of 3.71 mg/L test item was determined. An EC10 value of 11.0 mg/L test item an EC20 of 22.6 mg/L test item and an EC50 of 90.3 mg/L test item were calculated based on nominal concentrations.
No aborted brood or ephippia were observed in any treatment group.
The first offspring in control replicates was observed at day 11 after test start.

Reported statistics and error estimates:

Data were analysed using appropriate statistical methods, using ToxRat Professional 3.3 (2018). The NOEC/LOEC values and effect concentrations ECx values at 21 days for the reproductive output of the parent animals (number of offspring per introduced parent and number of offspring per survived parent), the development rate for reproduction and the intrinsic rate (r) of population increase were calculated. Additionally, LOEC/NOEC values for the mortality and age of first reproduction of the parent animals were determined.
The multiple sequentially-rejective Welch’s t-test after Bonferroni-Holm was used in relation to the cumulative number of offspring per introduced parent. The Williams multiple sequential t- test procedure was used in relation to the cumulative number of offspring per survived parent and for the intrinsic rate (r) of population increase.
Step-down Cochran-Armitage Test Procedure was used in relation to the mortality of parent Daphnia. Multiple Sequentially-rejective U-test After Bonferroni-Holm was used for the development rate for reproduction and the age of first reproduction. For the calculation of the ECx values (including 95 % confidence limits), a non-linear regression without weighting (estimated parameters of 3-parameters normal cumulative distribution function = CDF) was used. The linear regression method Probit analysis using linear max. likelihood regression was used for the endpoint mortality.

Results for reproduction and mortality of Daphnia magna

Treatment group	mg/L 3-Methylpyrazole, nominal
	control	1.23	3.71	11.1	33.3	100.0
21 days after application	Cumulative number of living offspring
Mean number per introduced parent	91.9	93.6	85.9	63.0	30.2	0.0
SD	6.40	5.19	8.79	9.76	6.29	0.0
CV (%)	7.0	5.5	10.2	15.5	20.8	n.d.
Mean number per survived parent	91.9	93.6	85.9	63.0	30.2	-
SD	6.40	5.19	8.79	9.76	6.29	-
CV (%)	7.0	5.5	10.2	15.5	20.8	-
	Reduction (%) in cumulative number of offspring per introduced parent^1relative to the control
	n.r.	-1.8	6.5	31.4 +	67.1 +	100.0 +
	Reduction (%) in cumulative number of offspring per survived parent^2relative to the control
	n.r.	-1.8	6.5 +	31.4 +	67.1 +	-
	Mortality of parent daphnids (%)^3
	0.0	0.0	0.0	0.0	0.0	100.0 +
	Mean intrinsic rate (r) of population increase
	0.293	0.293	0.291	0.262	0.215	5-
	Inhibition of intrinsic rate (r) relative to of population increase (%) the control					2
	n.r.	0.1	0.8	10.5 +	26.6 +	5-
	Age of first reproduction (Days)^4
	11.5	11.5	11.5	11.6	12.3	5-
	Development rate for reproduction
	0.087	0.087	0.087	0.086	0.082	5-
	Inhibition of development rate for reproduction (%)^4relative to the control
	n.r.	0.0	0.0	0.8	5.4	5-

SD = standard deviation, n.r. – not relevant, CV (%) = coefficient of variation

+ significantly different from control. Negative values for reduction of cumulative number of offspring indicate a higher reproduction than occurred for the control.

1 Multiple Sequentially-rejective Welch-t-test After Bonferroni-Holm

2 Williams multiple sequential t-test procedure, alpha = 0.05, one sided smaller

3 Step-down Cochran-Armitage Test Procedure, alpha = 0.05, one sided greater

4 Multiple Sequentially-rejective U-test After Bonferroni-Holm, alpha = 0.05

5 Treatments with no survivors were excluded from further analysis

Effects on reproduction of Daphnia magna: cumulative number of offspring and development rate

Effect concentration	mg/L 3-Methylpyrazole
	Mean cumulative number of offspring per introduced parent 21 days after application	Mean cumulative number of offspring per survived parent 21 days after application	Mortality of parents
NOEC
test item, nominal	3.71	1.23	33.3
LOEC
test item, nominal	11.1	3.71	100.0
EC10and 95 % confidence intervals (lower - upper)	EC10	EC10	EC10
test item, nominal	5.00 (3.80 - 6.60)	4.29 (3.02 - 6.11)	51.0n.d.
EC20and 95 % confidence intervals (lower - upper)	EC20	EC20	EC20
test item, nominal	7.91 (6.02 - 10.4)	7.20 (5.08 - 10.2)	53.2n.d.
EC50and 95 % confidence intervals (lower - upper)	EC50	EC50	EC50
test item, nominal	19.0 (13.6 - 26.4)	19.3 (12.5 - 29.6)	57.5n.d.

Calculations were conducted using unrounded values

n.d.: not determined due to mathematical reasons or inappropriate data

Effects on reproduction of Daphnia magna: mortality of parents, time until first reproduction and intrinsic rate of population increase

Effect concentration	mg/L 3-Methylpyrazole
	Development rate for reproduction	Age of first reproduction	Intrinsic rate of population increase*
NOEC test item, nominal	> 33.3	> 33.3	3.71
LOEC test item, nominal	> 33.3	> 33.3	11.1

Effects on reproduction of Daphnia magna: development rate, time until first reproduction and intrinsic rate of population increase

Effect concentration	mg/L 3-Methylpyrazole
	Development rate for reproduction	Age of first reproduction	Intrinsic rate of population increase*
EC10and 95 % confidence intervals (lower - upper)	EC10	EC10	EC10
test item, nominal	> 33.3	> 33.3	11.0 (8.14 - 14.8)
EC20and 95 % confidence intervals (lower - upper)	EC20	EC20	EC20
test item, nominal	> 33.3	> 33.3	22.6 (16.3 - 31.8)
EC50and 95 % confidence intervals (lower - upper)	EC50	EC50	EC50
test item, nominal	> 33.3	> 33.3	90.3 (50.3 - 157.9)

Calculations were conducted using unrounded values

n.d.: not determined due to mathematical reasons or inappropriate data

* The intrinsic rate is considered as a measure of population growth based on reproductive output and age-specific mortality. In steady state populations the intrinsic rate of population increase is zero, for growing populations it is positive and for shrinking populations it is negative

Validity criteria fulfilled:

yes

Conclusions:

A toxicity test was performed to assess the effects of the test item 3-Methylpyrazole on the reproductive output of Daphnia magna during 21 days of exposure.
The calculated study endpoints are based on nominal concentrations for the test item, all recoveries were within 80-120% of the nominal concentrations.
The lowest EC50 value was 19.0 mg/L test item for mean cumulative number of offspring per introduced parent.
The first offspring in control replicates was observed at day 11 after test start. No aborted brood or ephippia were observed in any treatment group.
100% mortality was observed at the highest test concentration of 100.0 mg/L test item.

Executive summary:

The primary objective of the test was to assess the effect of the test item on the reproductive output of Daphnia magna. The test duration was 21 days. At test end, the total number of living offspring produced was assessed. Data were analysed in order to determine the LOEC/NOEC values and to estimate concentrations that would cause a %-reduction in reproductive output (e.g. EC10, EC20 and EC50 values at 21 days), if possible. Furthermore, parental mortality was determined. Replicates were excluded if mortality was caused by technical reasons (mishandling or accident).

The test was performed using a semi-static test design (3 renewals per week). The study was conducted in accordance with the recommendations of the OECD Test Guideline No. 211 (2012).

Following findings were observed:

Mean cumulative number of offspring per introduced parent

The LOEC (lowest observed effect concentration) for mean cumulative number of offspring per introduced parent at 21 days after application was determined to be 11.1 mg/L test item. Hence, the NOEC (no observed effect concentration) was determined to be 3.71 mg/L test item, nominal. An EC10 value of 5.00 mg/L test item, an EC20 of 7.91 mg/L test item and an EC50 of 19.0 mg/L test item based on nominal concentrations, were calculated.

Mean cumulative number of offspring per survived parent

The LOEC (lowest observed effect concentration) for mean cumulative number of offspring per survived parent at 21 days after application was determined to be 3.71 mg/L test item. Hence, the NOEC (no observed effect concentration) was determined to be 1.23 mg/L test item, nominal. An EC10 value of 4.29 mg/L test item, an EC20 of 7.20 mg/L test item and an EC50 of 19.3 mg/L test item based on nominal concentrations, were calculated.

Mortality of parents

The control mortality of parent daphnids was 0%. Parental mortality occurred at the highest tested concentration of 100.0 mg/L test item, nominal at day 9-14 after test start (100%). The LOEC for mortality of parent animals was determined to be 100.0 mg/L test item, nominal. Hence, a NOEC of 33.3 mg/L test item, nominal was determined.

An EC10 value of 51.0 mg/L test item, nominal, an EC20 of 53.2 mg/L test item and an EC50 of 57.5 mg/L test item, based on nominal concentrations were calculated.

Age of first reproduction

The LOEC for the age of first reproduction was determined to be higher than 33.3 mg/L test item, nominal Hence, a NOEC of higher or equal 33.3 mg/L test item based on nominal concentrations was determined. EC10, EC20 and EC50 values were higher than 33.3 mg/L test item.

Development rate for reproduction

The LOEC for the development rate for reproduction was determined to be higher than 33.3 mg/L test item, nominal Hence, a NOEC of higher or equal 33.3 mg/L test item based on nominal concentrations was determined. EC10 EC20 and EC50 values of higher than 33.3 mg/L test item, based on nominal concentrations were calculated.

Intrinsic rate (r) of population increase

The LOEC for intrinsic rate of population increase was determined to be 11.1 mg/L test item. Hence, a NOEC of 3.71 mg/L test item was determined. An EC10 value of 11.0 mg/L test item an EC20 of 22.6 mg/L test item and an EC50 of 90.3 mg/L test item were calculated based on nominal concentrations.

No aborted brood or ephippia were observed in any treatment group.

The first offspring in control replicates was observed at day 11 after test start.

Description of key information

The primary objective of the test was to assess the effect of the test item on the reproductive output of Daphnia magna. The test duration was 21 days. At test end, the total number of living offspring produced was assessed. Data were analysed in order to determine the LOEC/NOEC values and to estimate concentrations that would cause a %-reduction in reproductive output (e.g. EC10, EC20 and EC50 values at 21 days), if possible. Furthermore, parental mortality was determined. Replicates were excluded if mortality was caused by technical reasons (mishandling or accident).

The test was performed using a semi-static test design (3 renewals per week). The study was conducted in accordance with the recommendations of the OECD Test Guideline No. 211 (2012).

Following findings were observed:

Mean cumulative number of offspring per introduced parent

The LOEC (lowest observed effect concentration) for mean cumulative number of offspring per introduced parent at 21 days after application was determined to be 11.1 mg/L test item. Hence, the NOEC (no observed effect concentration) was determined to be 3.71 mg/L test item, nominal. An EC10 value of 5.00 mg/L test item, an EC20 of 7.91 mg/L test item and an EC50 of 19.0 mg/L test item based on nominal concentrations, were calculated.

Mean cumulative number of offspring per survived parent

The LOEC (lowest observed effect concentration) for mean cumulative number of offspring per survived parent at 21 days after application was determined to be 3.71 mg/L test item. Hence, the NOEC (no observed effect concentration) was determined to be 1.23 mg/L test item, nominal. An EC10 value of 4.29 mg/L test item, an EC20 of 7.20 mg/L test item and an EC50 of 19.3 mg/L test item based on nominal concentrations, were calculated.

Mortality of parents

The control mortality of parent daphnids was 0%. Parental mortality occurred at the highest tested concentration of 100.0 mg/L test item, nominal at day 9-14 after test start (100%). The LOEC for mortality of parent animals was determined to be 100.0 mg/L test item, nominal. Hence, a NOEC of 33.3 mg/L test item, nominal was determined.

 

An EC10 value of 51.0 mg/L test item, nominal, an EC20 of 53.2 mg/L test item and an EC50 of 57.5 mg/L test item, based on nominal concentrations were calculated.

 

Age of first reproduction

The LOEC for the age of first reproduction was determined to be higher than 33.3 mg/L test item, nominal Hence, a NOEC of higher or equal 33.3 mg/L test item based on nominal concentrations was determined. EC10, EC20 and EC50 values were higher than 33.3 mg/L test item.

Development rate for reproduction

The LOEC for the development rate for reproduction was determined to be higher than 33.3 mg/L test item, nominal Hence, a NOEC of higher or equal 33.3 mg/L test item based on nominal concentrations was determined. EC10 EC20 and EC50 values of higher than 33.3 mg/L test item, based on nominal concentrations were calculated.

Intrinsic rate (r) of population increase

The LOEC for intrinsic rate of population increase was determined to be 11.1 mg/L test item. Hence, a NOEC of 3.71 mg/L test item was determined. An EC10 value of 11.0 mg/L test item an EC20 of 22.6 mg/L test item and an EC50 of 90.3 mg/L test item were calculated based on nominal concentrations.

No aborted brood or ephippia were observed in any treatment group.

The first offspring in control replicates was observed at day 11 after test start.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Remarks:

based on mean cumulative number of offspring per survived parent 21 days after application

Effect concentration:

1.23 mg/L