DL-thyroxin

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 January - 29 April 2020

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: LEVOTHYROXINE ACID
Batch No.: B486066
Expiry date:27 August 2020
Storage condition: at room temperature, protected from light
Appearance:white slightly creme, powder

Analytical monitoring:

yes

Details on sampling:

For determination of the test item concentration, four replicate samples (5 mL per replicate) were taken from the test item solution and from the control at the start and at the end of the test.

Vehicle:

no

Details on test solutions:

The test solution used in the test was prepared by mechanical dispersion without using of any solubilising agent. The test item is poorly water soluble material. For preparation of the test solution a supersaturated solution (100 mg/L nominal loading) was prepared by dispersing/dissolving an amount of 0.0307 g test item in 307 mL dilution water (ISO Medium). This solution was agitated by orbital shaker (~300 rpm) for a period of at least 2 days (approx. 4 days in this test) and then the non-dissolved test material was separated by filtration through a membrane filter (0.45 μm) in order to obtain the saturated test solution (corresponding to 100 mg/L nominal concentration). Untreated control ran parallel in the test. After the formulation procedure the test animals were immediately introduced into test solution.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species and strain: Daphnia magna (Straus)
Origin: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection)
Breeding: The Daphnia are bred in the Laboratory of TOXI-COOP ZRT.
Number of animals: Twenty animals in each group (concentrations and untreated control), divided into 4 concurrent batches (5 animals per batch).
Age of animals: Less than 24 h old at the beginning of the test.
Sex: Female
Animal health: Apparently healthy animals were used in this test with a known history
Acclimatization: Test animals were bred under similar (or the same) conditions as that used during the exposure period (holding water, temperature, background colour etc.), therefore additional acclimatisation before the test was not necessary. Brood daphnids were maintained in dilution water at the test temperature for at least 48 hours prior to the start of the test.
Food and Feeding: The Daphnia were fed with concentrated algal suspension of Raphidocelis subcapitata during the holding. Test animals were not fed during the exposure.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

The reconstituted water (ISO medium) has an approximate theoretical total hardness of 249 mg/L (as CaCO3).

Test temperature:

The test temperature was in the range of 20.9 – 21.1°C measured in the test vessels during the test. The additionally measured ambient temperature within the climate chamber was between 20.4 – 21.4°C.

pH:

The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test group. The pH was in the range of 7.73 – 8.16 during the test.

Dissolved oxygen:

The dissolved oxygen concentration was in the range of 8.02 – 8.36 mg/L during the test.

Nominal and measured concentrations:

During the experiment the test item was tested at aquatic saturation (equivalent to 100 mg/L nominal concentration). A concurrent control ran.
The mean measured concentration of the test item was 0.40 mg/L at the start and 0.26 mg/L at the end of the test.
Exposure concentration of the test item was calculated as the geometric mean of the measured start and end concentrations and was determined to be 0.32 mg/L.

Details on test conditions:

Twenty animals, divided into four groups (glass beaker; volume app. 50 mL) of five animals each were exposed to the test item or included as control over a period of 48 hours in a static test. The test animals were not fed and the test vessels were not aerated during the test. Each test vessel contained approximately 40 mL of test solution and was uniquely identified with study code, test group and replicate. Daphnia were observed for immobility or mortality by visual observation after 24 and 48 hours of exposure. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile.

Reference substance (positive control):

yes

Remarks:

Name: Potassium dichromate (K2Cr2O7) CAS No.: 7778-50-9 Batch No.: K50498364 Expiry Date: 31 July 2023 Supplier: MERCK

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: saturation equivalent to > 100 mg/L nominal

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

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

> 0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

There was no immobilisation observed in twenty daphnids exposed to either test item treated or control group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected.Accordingly, the 48-h NOEC was determined as the saturation concentration (equivalent to 100 mg/L nominal concentration; 0.32 mg/L measured).The 48-h EC50 and LOEC were determined to be higher than saturation (equivalent to > 100 mg/L nominal concentration; > 0.32 mg/L measured).

Results with reference substance (positive control):

The immobilisation rate of the introduced Daphnia should not exceed 10 % in the control group.
The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels. If the percentage of immobilisation exceeds 10 % at the end of the test, a full study should be conducted.

Validity criteria fulfilled:

yes

Conclusions:

In this 48-hour static acute toxicity test with Daphnia magnathe test item LEVOTHYROXINE ACID had no toxic effect on Daphnia at aquatic saturation (measured value: 0.32 mg/L; equivalent to 100 mg/L nominal concentration).
Accordingly, the 48-h EC50 value was determined to be > 0.32 mg/L (equivalent to > 100 mg/L nominal concentration).
The 48-h NOEC was determined to be 0.32 mg/L (equivalent to 100 mg/L nominal concentration). All validity criteria were met.

Executive summary:

Test design

Test item:LEVOTHYROXINE ACID(Batch No.: B486066)

Test species:Daphniamagna(Straus)
Dilution water:ISO medium, prepared in the laboratory of TOXI-COOP ZRT.

Concentrations
Limit test; using a single concentration at saturation (corresponding to 100 mg/L nominal concentration); concurrent control ran.The exposure concentration of the saturated test solution was analytically determined to be 0.32 mg/L (calculatedas the geometric mean of measured start and endconcentrations).
Test design:
Twenty animals, divided into four groups (glass beaker; volume app. 50 mL) of five animals each were exposed to the test concentration or ran as control for 48 hours in a static test.The immobility or mortality of the Daphnia wasdetermined by visual observation after 24 and 48 hours of exposure.

Analytics:
Concentration of the test item were determined using HPLC-MSmethod at the start and at the end of the test.

Endpoints:EC10, EC20, EC50, NOEC and LOEC for 48h test period.

Statistics:Statistical analysis was not necessary; endpoints were determined directly from the raw data.

Results

Validity:All validity criteria were met and therefore the study can be considered as valid.

Analytical results:The mean measured test item concentration of the saturated test solution was 0.40 mg/L at the start and 0.26 mg/L at the end of the test.

Results:There was no immobilisation observed in twenty daphnids exposed to the test item treated and control group. Further no abnormal behaviour or appearance of test animals was noted.

The biological endpoints are summarised below in Table 1.

 

Table 1: Summary of the Biological Endpoints

Endpoint	Concentration [mg/L] based on nominal concentrations
48 h-EC10	> 0.32 mg/L (equivalent to > 100mg/L nominal)
48 h-EC20	> 0.32 mg/L (equivalent to > 100mg/L nominal)
48 h-EC50	> 0.32 mg/L (equivalent to > 100mg/L nominal)
48 h-NOEC	0.32 mg/L (equivalent to > 100mg/L nominal)
48 h-LOEC	> 0.32 mg/L (equivalent to > 100mg/L nominal)

 

Description of key information

In the 48-hour static acute toxicity test with Daphnia magnathe test item LEVOTHYROXINE ACID had no toxic effect on Daphnia at aquatic saturation (measured value: 0.32 mg/L; equivalent to 100 mg/L nominal concentration).
Accordingly, the 48-h EC50 value was determined to be > 0.32 mg/L (equivalent to > 100 mg/L nominal concentration).
The 48-h NOEC was determined to be 0.32 mg/L (equivalent to 100 mg/L nominal concentration). All validity criteria were met.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L