Pirimiphos-methyl

Endocrine disrupter testing in aquatic vertebrates – in vivo

Administrative data

Endpoint:

fish early-life stage: reproduction, (sub)lethal effects

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start: 11 November 2021, experimental completion: 27 June 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD TG 230 (21-day Fish Assay: A Short Term Screening for Oestrogenic and Androgenic Activity, and Aromatase Inhibition)

Version / remarks:

2009

Deviations:

yes

Remarks:

Test fish were slightly older than recommended in the guideline. Total hardness and alkalinity were not measured in one vessel of the highest test concentration. These deviations are not expected to have an impact on the acceptability of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Pirimiphos-methyl

EC Number:

249-528-5

EC Name:

Pirimiphos-methyl

Cas Number:

29232-93-7

Molecular formula:

C11H20N3O3PS

IUPAC Name:

O-2-(diethylamino)-6-methylpyrimidin-4-yl O,O-dimethyl phosphorothioate

Test material form:

liquid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The flow through system was started two days before introduction of the fish for equilibration of the system. For this, application solutions were freshly prepared two days before introduction of the fish into the test media.
For the determination of the actual test item concentrations and stability of the test item in the application solutions single samples of 3 mL were taken from the freshly prepared application solutions at the start of the equilibration period, and Days 6 and 14.
From the aged application solutions, samples were taken on Days 6, 14 and 21 at the end of the application solution renewal periods (7- and 8-days stability controls).
For the determination of the actual test item concentration in the exposure tanks, duplicate samples (10 mL) of all four replicates of the control, the solvent control and all test concentrations were taken one day before start of exposure, at the start of the exposure and at Days 7 and 14 and at the end of the exposure.
All samples were stored deep-frozen (at about –20 °C) immediately after sampling until analysis. In pre-experiments for investigation of the storage stability of the samples, the test item proved to be stable under these storage conditions.
From the application solutions of all three test concentrations, one sample from a freshly prepared and aged application solutions was analysed from an 8-days stability control (Day 6 to Day 14).
From the test medium samples of all three test concentrations and from the control and solvent control, one of the duplicate samples from all four replicates was analysed from the samplings at Day 0, 7, 14 and 21.

Test solutions

Vehicle:

yes

Remarks:

N,N-dimethylformamide

Details on test solutions:

Flow-through System:
For continuous dosing of the test item into test water an automated flow-through dosing system was used (Pequitec MSR System, 4852 Rothrist, Switzerland). The dosing system consisted of five individual dosing units which were used for test medium supply for the three test concentrations and the water supply of the control and the solvent control.
Solubility tests and a dosing pre-experiment were performed to select appropriate dosing parameters (e.g. concentration of application solution, stirring time in the mixing vessel, test medium exchange rate) and to ensure a constant exposure concentration of the test item during the test period.
For the dosing of the test item into test water, concentrated application solutions (test item dissolved in N,N-dimethylformamide, DMF) of the test item were prepared for each test concentration. The dosing system was started two days before introduction of the fish for equilibration of the system (two-day pre-dosing period) and correct dosing was confirmed analytically.
Preparation of Application Solutions:
For the preparation of the application solutions used for the preparation of the test media, the application solution used for the dosage of the highest test concentration was prepared by completely dissolving 1562.5 mg of the test item (effective range: 1562.59 – 1564.11 mg) in 125 mL DMF (first application solution) by stirring for 15 minutes at room temperature. This application solution was diluted with DMF to prepare the application solutions used for dosage of the lower test concentrations, i.e., 20 mL of the application solution for the highest test concentration was filled up to 100 mL with DMF, resulting in an application solution with 50.0 μg/L test item. In order to prepare the application solution for the lowest test item concentration, 4 mL of the highest application solution was filled up to 100 mL with DMF, resulting in an application solution with 10.0 μg/L test item.
The application solutions were freshly prepared three times during the pre-dosing period and the exposure period.
The application solutions were continuously stirred and protected from light during their use. The stability of the test item in the application solutions was examined in analytical pre-experiments and was confirmed by analytical measurements within the main test.

Test organisms

Aquatic vertebrate type:

fish

Test organisms (species):

Pimephales promelas

Details on test organisms:

Test fish: fathead minnow (Pimephales promelas, Rafinesque 1820).
Supplier: OSAGE Catfisheries, Inc., Osage Beach, Missouri, USA
Mean wet weight: 1.8 g (1.5 - 2.2 g) for females and 3.2 g (2.6 - 3.8 g) for males
Sexually dimorphic, reproductively active fish (beginning of their reproductive phase) were used. The fish originated from one and the same breeding batch and were held separately by sex until start of the pre-exposure period. The age of the fish at the start of the pre-exposure period was approximately 8 months. Before the start of the pre-exposure period, a subsample of this batch was used to determine the arithmetic mean wet weight of fish per sex. Based on this information, fish with a wet weight within ± 20% (if possible) of the arithmetic mean wet weight of this breeding batch were selected for the start of the pre-exposure period. The fish used for determination of the arithmetic mean were not used. During the pre-exposure period, the fish held in spawning groups, consisting of one male and two females, were regularly spawning and groups selected for the exposure showed 5-10 spawns per group and 28 to 52 eggs/female/day.
State: all fish were healthy and in a good state, nor mortality occurred n at least three weeks before the experimental start.
Acclimatisation: for at least two weeks prior to test start
Feed: commercial dry food (TETRA MIN Hauptfutter, TETRA-Werke, 49324 Melle, Germany) given daily ad libitum in at least three portions on working days and in at one or two portions on weekends. Food was withheld from the fish for 24 hours prior to sampling.
Faeces and uneaten food were removed regularly from the test vessels.

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test conditions

Hardness:

125 - 130 mg/L (as CaCO3)

Test temperature:

24.1 to 24.7 °C

pH:

7.0 to 7.1

Dissolved oxygen:

During the test period, the lowest dissolved oxygen concentration measured in a single replicate of the control and solvent control and at the test concentrations was 6.6 mg/L, corresponding to an oxygen saturation of at least 82%.

Salinity:

Not applicable

Conductivity:

342 to 357 µS/cm2

Nominal and measured concentrations:

Nominal: 10, 50 and 250 µg/L
Time-weighted average measured: 7.9, 42 and 215 µg/L

Details on test conditions:

Test 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 x 2 H2O at 147 mg/L, MgSO4 x 7 H2O at 61.5 mg/L, NaHCO3 at 32.5 mg/L, KCl at 2.9 mg/L.

Preparation of Test Media:
Two days before introduction of the fish, the automatic dosing system was started for equilibration of the dosing system.
In the individual dosing units, the test medium was prepared batch wise by mixing defined volumes of application solution in test water by intensive stirring. This batch of test medium was then distributed to the four replicate test vessels.
A cycle for test medium preparation in the dosing units started by filling a mono-block glass mixing chamber with 3000 mL of test water by use of a peristaltic pump. During the filling process, 60 μL application solution were dosed into the test water by use of a syringe pump. Test water and application solution were stirred by a magnetic stirrer for 2 minutes to ensure that the aqueous application solutions and test water were properly mixed. Test water and application solution formed the test medium with the corresponding test concentration.
After stirring and by opening a magnetic valve, the test medium was released from the mixing chamber to the four replicates by hydrostatic pressure. The test medium was split into four and directed to the different replicates by a Teflon tube. In this way, the test medium flow was the same for all four replicates.
After all test medium in the mixing chamber had been distributed, a new test medium preparation cycle started.
The frequency of test medium preparation cycles was 8 minutes, i.e., 7.5 cycles per hour (= 180 cycles per day). Per cycle, 3-liter medium were prepared in the mixing vessel.
Thus, in total 540-liter test medium per day were prepared for each treatment. This volume was distributed to the four replicate aquaria with 23-liter volume each. This test medium flow resulted in a 6-fold theoretical water exchange rate per day in each of the four replicate aquaria. Test water with 20 μL/L DMF flowed at the same rate into the replicates of the solvent control. Test water without any addition of test item flowed at the same rate into the replicates of the control.
The dosing system was visually inspected at least once a day to ensure flows and mixing were working properly. The correct function was inspected each working day by volumetric measurements (filling level of the test water in the mixing chamber) and gravimetric measurements (amount of application solution dosed into the mixing chamber). The application solutions and test media were observed for precipitation on a daily basis.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

215 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Nuptial Tubercle Scores and vitellogenin

Effect concentrations 2

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

215 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

gonadal histology

Details on results:

Mortality and abnormalities
The survival of test fish in the control and solvent control was 100% in the course of the study, and the validity criterion stated in the guidance of at least 90% was met. The survival of test fish in the treated groups was 100%. During the exposure period, no visible abnormalities and no signs of general toxicity were observed in any of the control or treated groups. All fish appeared healthy and showed typical behaviour. Male and female fish had couloration and couloration patterns typical of the respective sex. The dorsal nape pad was clearly expressed in all male fish. Male fish showed territorial behaviour and mating behaviour, which was considered typical of fathead minnow. The NOEC for mortality and visible abnormalities was 215 µg/L.
Body length and weight
The measured body length and body wet weight of female and male fish at the end of the test was evenly distributed among the different test groups and replicates. The CV of body length and body wet weight of males and females for all fish used in the test was less than 20%.
Spawning
Spawning activity (i.e. presence and absence of eggs on a given day) was regular in all control groups and treated groups, showing that test fish used in the study were in the reproductive stage and actively spawning at regular intervals throughout the study.
Nuptial tubercles
The number of tubercles for the male fish of the control and solvent control, were in the range of 20.0 ± 1.87 and 20.5 ± 2.89, respectively. No significant differences between control and solvent control were found. The number of tubercles was 20.1 ± 1.65, 21.6 ± 2.29 and 19.0 ± 2.12 in the groups exposed to 7.9, 42 and 215 μg/L (mean measured), respectively. The statistical examination of the total number of tubercles of the males exposed to the different test item concentrations showed no statistically significant differences to the solvent control (Dunnett’s t-test after Bonferroni-Holm, two-sided α = 0.05).
The tubercle score for the male fish of the control and solvent control, were in the range of 32.8 ± 2.72 and 33.6 ± 3.07, respectively. No significant differences between control and solvent control were found. The tubercles scores were 32.6 ± 3.15, 34.6 ± 2.10 and 32.5 ± 2.80 in the groups exposed to 7.9, 42 and 215 μg/L (mean measured), respectively, and no statistically significant differences to the solvent control were found (Dunnett’s t-test, two-sided α = 0.05).
Thus, the expression or the appearance of tubercles in male fish was not affected and the NOEC was 215 µg/L.
No nuptial tubercles were observed in female fish in control or treatment groups.
Vitellogenin
In male fish, plasma vitellogenin levels of 7.1 ± 12.3 μg/mL and 29.2 ± 42.3 μg/mL were found in the control and solvent control, respectively. The difference was not found to be statistically significant. Mean plasma vitellogenin levels were 9.48 ± 16.6, 23.8 ± 43.3 and 14.4 ± 12.5 in male fish exposed to 7.9, 42 and 215 μg/L (mean measured), respectively. In males no statistically significant increase in vitellogenin levels was found at any test concentration.
In female fish, plasma vitellogenin levels of 5398 ± 1054 μg/mL and 4255 ± 918 μg/mL were found in the control and solvent control, respectively. The difference was not found to be statistically significant. Mean plasma vitellogenin levels were 4994 ± 1186, 4486 ± 497 and 5013 ± 1676 in female fish exposed to 7.9, 42 and 215 μg/L (mean measured pirimiphos-methyl), respectively. In females no statistically significant change in vitellogenin levels was found at any test concentration.
Histopathology
Under the conditions of this study, no treatment-related histopathological changes in the gonads or gonadal staging (testis and ovary) have been observed after exposure to pirimiphos-methyl.

Results with reference substance (positive control):

Not applicable

Any other information on results incl. tables

The test was considered to be valid as the following criteria given by the test guideline were fulfilled:
The survival of the test fish in the control should be at least 90% at the end of the exposure period. In the present study the survival of the test fish in the control and solvent control was 100%.
The dissolved oxygen concentration in the test media should be at least 60% of the air saturation value throughout the test. In the present study minimum oxygen concentration measured in a replicate was 82%.
The water temperature should not differ by more than ±1.5 °C between the test vessels at any one time during the exposure period and be maintained within a range of 25.0 ±2 °C. In the present study the temperature measured in the individual replicates was in the range of 24.1 to 24.7 °C.
The coefficients of variation (CV) for the individual measurements in the aqueous exposure media were 3.6, 4.8 and 3.2 % for the nominal test item concentrations of 10, 50 and 250 μg/L, respectively. The test concentrations of the test substance have been satisfactorily maintained within ± 20% of the mean measured values and were within 20 % of nominal test concentrations based on active ingredient.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The study did not provide evidence that the substance has a potential to interact with the hypothalamus-pituitary-gonadal (HPG) axis of spawning fish of the species fathead minnow.

Executive summary:

A short-term study was performed under GLP to OECD TG 230 to screen for oestrogenic and androgenic effects and aromatase inhibition as primary indicators of potential endocrine effects in spawning freshwater fish fathead minnow (Pimephales promelas). At the start of exposure, spawning groups of sexually mature male and female fathead minnows (2 male and 4 female) were distributed to each of the four replicates of each treatment. Survival and observations on behaviour and spawning activity were recorded daily during the exposure period. Body length and body wet weight of the test fish were measured at test end. Additional endpoints were captured and used as indicators of potential endocrine activity, namely secondary sexual characteristics of the fish, vitellogenin concentration in blood plasma and gonad histology (testes and ovaries) after the exposure period of 21 days.
The test was run under flow-through conditions with a six-fold theoretical water exchange rate in the test vessels. For the continuous dosage of the test item in test water, concentrated application solutions in DMF were used (20 μL/L N,N-dimethylformamide, DMF). The nominal concentrations of the test item were 10, 50 and 250 μg/L. These were confirmed by analysis with a suitable HPLC-MS/MS method and mean measured concentrations were 9.23, 46.2 and 231 µg/L. Additionally, a control group and a solvent control group were tested in parallel.
The test was considered valid as the following criteria given by the test guidelines were fulfilled: Survival of the test fish in the control was 100%, therefore, fulfilling the criteria of a survival rate of at least 90%. The dissolved oxygen saturation was at minimum at 82% (validity criteria: at least 60% of the air saturation value) and water temperature was in the range of 24.1 to 24.7 °C (validity criteria: 25 ± 2 °C) and did not differ by more than ± 1.5 °C between the test vessels at any one time during the exposure period.
During the test, no mortality and no visible abnormalities were observed in the control and test groups. Mating behaviour of fish was normal in all control and test groups, and regular spawning of females was seen in all replicates over the test period of 21 days. Exposure of the test fish to the substance did not result in a significant increase of vitellogenin levles in males or significant change of vitellogenin levels in female fish compared to the control groups. Exposure to the substance had no effect on the number of nuptial tubercles in male fish exposed to the substance. Growth endpoints (total body length and wet body weight) provided no evidence of general toxic effects of the test substance. Based on histological examinations, the test substance did not have any effect on the endocrine system at all tested concentrations. There were any histopathological lesions in the testes or ovaries, nor any changes of the gonadal staging, which could be attributed to exposure to the test substance.
Overall, the study gave no evidence that the substance exhibits endocrine activity in spawning fish exposed to concentrations of up to 215 µg/L over a period of 21 days.