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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-05-04 to 2018-09-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD, EU Guideline Study: GLP
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Qualifier:
according to
Guideline:
other: OECD No 23; Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Name: TMPMP
CAS-No.: 33007-83-9
Batch No.: 0061487
Purity: 97.6 % according to certificate of analysis
CoA Reference No.: P292/2016
Manufacturing Date: December 21, 2017
Aggregate State at Room Temperature: Liquid
Colour: Colourless
Density: 1.21 g/cm³ according to MSDS
Expiry Date: December 20, 2018
Storage Conditions at Test Facility:At 4 +/- 4 °C, in the dark
Analytical monitoring:
yes
Remarks:
LC-MS/MS
Details on sampling:
Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test and at day 3.
For the determination of the stability of the test item under the test conditions, respectively the maintenance of the test item concentrations during the test period, samples were taken in duplicate out of all test media and the control at day1 and at the end of the test (96 hours) from the approximate centre of the aquaria.
However, from the test concentration of 1.235 mg test item/L duplicate samples were taken at day 2, respectively, because all fish were dead.
The samples were diluted by a factor of two with acetonitrile.
Additional samples of the control and of the dilution solvent were taken at each sampling without any sample treatment.
Vehicle:
no
Details on test solutions:
Due to the low limit of water solubility of the test item, a filtrate of nominal 100 mg/L was prepared. Dilutions of 1:3, 1:9, 1:27, 1:81, 1:243 and 1:729 of this filtrate were used as test concentrations. Additionally, a control was tested in parallel.
The following geometric mean measured concentrations of the test item correspond to the above mentioned dilutions:
9.92, 3.09, 1.02, 0.335, 0.0728 and 0.0294 mg test item/L, and a control.
In the control, test water was used without addition of the test item.
Supersaturated stock solutions of nominal 100 mg test item/L were prepared by suspending 520 mg test item in 5200 mL test water. Each stock solution was stirred for 48 hours at room temperature in the dark to dissolve as much test item as possible. Then non-dissolved fractions of the test item were separated from the test medium by membrane filtration (0.45 µm cellulose acetate filter). Adequate volumes of this filtrate were mixed into test water to obtain the desired dilutions of 1:3, 1:9, 1:27, 1:81, 1:243 and 1:729. These dilutions were used as test concentrations
The test media were prepared just before introduction of the test fish (= start of the test and test medium renewal on day 1, 2 and 3).
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Species: Rainbow trout (Oncorhynchus mykiss)
Size and Weight: Juveniles;
The mean body length of the fish* in the test was 4.89 cm ± 0.4 cm (Mean ± SD), the mean body wet weight 1.20 g ± 0.3 g (Mean ± SD).
* 10 fish from the test fish batch were measured before the start of the test
Sex: Male and female
Origin: The test fish were obtained from Forellenzuchtbetrieb Störk, 88348 Bad Saulgau, Germany Holding Conditions: All fish were obtained and held in the laboratory for at least 12 days before the start of the test. They were held in water of the quality to be used in the test for at least seven days immediately before testing under the following conditions:
Light: 16 hours photoperiod daily
Temperature: 13 - 17 °C
Oxygen concentration: at least 80 % of the air saturation value
Feeding: three times per week or daily until 24 hours before the test was started
During the last 7 days prior to the start of the test no fish (0 %) died in the test fish batch. Therefore the mortalities in the fish batch were below 5 % and the fish batch was accepted.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
no
Hardness:
2.5 mmol/L (= 250.0 mg/L) as CaCO3
Test temperature:
14 - 15 °C
pH:
7.5 to 7.9
Dissolved oxygen:
94 to 101 %
Nominal and measured concentrations:
Due to the low limit of water solubility of the test item, a filtrate of nominal 100 mg/L was prepared. Dilutions of 1:3, 1:9, 1:27, 1:81, 1:243 and 1:729 of this filtrate were used as test concentrations. Additionally, a control was tested in parallel.
The following geometric mean measured concentrations of the test item correspond to the above mentioned dilutions:
9.92, 3.09, 1.02, 0.335, 0.0728 and 0.0294 mg test item/L, and a control.
Details on test conditions:
Test Environment: Controlled environmental room
Measurement of pH-Values, Dissolved Oxygen and Water Temperature:
The water temperature, pH-values and the dissolved oxygen concentrations were determined daily in the freshly prepared and aged test media of each treatment group.
Water Temperature: 14 to 15 °C pH-Values of the Test Water: 7.5 to 7.9
Dissolved Oxygen Concentrations: 94 to 101 % of the air saturation value Light Regime: 16 h light : 8 h dark; 30 min dawn/dusk period was provided
Light Intensity: 650 to 760 lux Aeration of the Test Water: The test media were slightly aerated during the test.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.073 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
0.335 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
0.156 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
In the control and at the concentrations up to 0.0728 mg test item/L, all fish survived until the end of the experiment and no signs of intoxication occurred. At 0.335 to 9.92 mg test item/L, all fish died until the end of the test.
Reported statistics and error estimates:
The LC50, LC20 and LC10 at the observation times were calculated by Probit analysis. The NOEC, the LOEC, the LC0 and the LC100 were determined directly from the raw data.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.
Validity criteria fulfilled:
yes
Remarks:
In the control no fish died during the test. The dissolved oxygen concentration in the test media did not fall below 94 % of air saturation value during the test.
Conclusions:
The toxic effect of the test item TMPMP to Rainbow Trout (Oncorhynchus mykiss) was assessed in a semi-static concentration-response test. Based on the test results the 96-hour LC50 was determined to be 0.156 mg test item/L based on geometric mean measured concentrations. The NOEC was determined to be 0.0728 mg test item/L also based on geometric mean measured concentrations.
The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to geometric mean concentrations, since the test item concentrations were not within ± 20 % of the measured initial concentrations during the test.
Executive summary:

Title: TMPMP: Acute Toxicity to Rainbow Trout (Oncorhynchus mykiss) in a 96-hour Semi-Static Test

Purpose:

The purpose of this study was to evaluate the acute toxicity of the test item TMPMP to fish. For this purpose, juvenile Rainbow Trout were exposed in a semi-static test to various concentrations under defined conditions for 96 hours. The recorded effects were the mortality and sublethal effects on the fish. The test method of application and the test system are recommended by the test guidelines and Rainbow Trout is one of the recommended test species. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

Guidelines:

-Commission Regulation (EC) No 440/2008, Annex, Part C, C.1.: “Acute Toxicity for Fish”, Official Journal of the European Union, May 30, 2008

-OECD Guideline for Testing of Chemicals, Section 2, No. 203, “Fish, Acute Toxicity Test”, adopted July 17, 1992

-OECD Series on Testing and Assessment, No. 23, “Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures”, December 15, 2000

-SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

Materials and Methods

Test Item: TMPMP; batch no.: 0061487; purity: 97.6 %, according to certificate of analysis

Test Species: Juvenile Rainbow Trout (Oncorhynchus mykiss) mean length: 4.89 cm ± 0.4 cm; source: Forellenzuchtbetrieb Störk, 88348 Bad Saulgau, Germany

Test Design:

This study encompassed 7 treatment groups (6 dose rates of the test item and a control) each containing 7 individuals. The acute toxicity to unfed juvenile Rainbow Trout was determined in an aerated, semi-static, 96-hour test. The test fish were observed at test start and after approximately 2, 24, 48, 72 and 96 hours test duration for sublethal effects and mortality. Dead fish were removed at least once daily and discarded.

Endpoints: NOEC after 96 h, LOEC after 96 h; LC0,50,100: lethal concentration producing 0, 50and 100 % mortality after 96 h of exposure.

Test Concentrations:

Due to the low limit of water solubility of the test item, a filtrate of nominal 100 mg/L was prepared. Dilutions of 1:3, 1:9, 1:27, 1:81, 1:243 and 1:729 of this filtrate were used as test concentrations. Additionally, a control was tested in parallel.

The following geometric mean measured concentrations of the test item correspond to the above mentioned dilutions:

9.92, 3.09, 1.02, 0.335, 0.0728 and 0.0294 mg test item/L, and a control.

Test Conditions: Water temperature: 14 to 15 °C; pH value: 7.5 to 7.9; dissolved oxygen concentration: 94 to 101 % of the air saturation value; photoperiod: 16 h light - 8 h dark; light intensity: 650 to 760 lux and thus were within the ranges requested by guideline OECD 203.

Results:

Biological test results:

Sublethal Effects: In the control and at the concentrations up to 0.0728 mg test item/L, all fish survived until the end of the experiment and no signs of intoxication occurred. At 0.335 to 9.92 mg test item/L, all fish died until the end of the test.

96-hour LC50: 0.156 mg test item/L

95 % Confidence Interval: n.d.

96-hour LC20: 0.140 mg test item/L

95 % Confidence Interval: n.d.

96-hour LC10: 0.132 mg test item/L

95 % Confidence Interval: n.d.

96-hour LC0: 0.0728 mg test item/L

96-hour LC100: 0.335 mg test item/L

96-hour NOEC: 0.0728 mg test item/L

96-hour LOEC: 0.335 mg test item/L

Analytical test results:

The quantification of the test item TMPMP in the test samples was performed using liquid chromatography with MS/MS.

The concentrations of the test item were determined in the duplicate test media samples from all test concentrations and the duplicate control samples from all sampling times. The geometric mean measured values are

9.92 mg test item/L in the 1:3 dilution of filtrate

3.09 mg test item/L in the 1:9 dilution of filtrate

1.02 mg test item/L in the 1:27 dilution of filtrate

0.335 mg test item/L in the 1:81 dilution of filtrate

0.0728 mg test item/L in the 1:243 dilution of filtrate

0.0294 mg test item/L in the 1:729 dilution of filtrate

Conclusion:

The toxic effect of the test item TMPMP to Rainbow Trout (Oncorhynchus mykiss) was assessed in a semi-static concentration-response test. Based on the test results the 96-hour LC50 was determined to be 0.156 mg test item/L based on geometric mean measured concentrations. The NOEC was determined to be 0.0728 mg test item/L also based on geometric mean measured concentrations. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to geometric mean concentrations, since the test item concentrations were not within ± 20 % of the measured initial concentrations during the test.

This study is classified acceptable and satisfies the guideline requirements for fish acute test studies.

Description of key information

The measured 96-h LC50 of TMPMP for freshwater fish is 0.156 mg/L

Key value for chemical safety assessment

LC50 for freshwater fish:
0.156 mg/L

Additional information