DL-thyroxin

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 January 2020 - 30 March 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Annex 5 corrected: 28 July 2011

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Toxic effects were not observed during the preliminary test, therefore only one test concentration at saturation (equivalent to 100 mg/L nominal concentration) and one control group were tested in a limit test.The exposure concentration of the saturated test solution was analytically determined to be 1.04 mg/L (calculated as the geometric mean of measured start and end concentrations).

Vehicle:

no

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

Culture temperature was checked at the beginning of the study and each day thereafter in a flask filled with water, in the climatic chamber. In addition temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was between 22.3 and 22.7 °C measured in the flask and in the range of 21.2 – 23.2 °C measured within the climate chamber.

pH:

The pH was measured in each test group at the start (before test solutions had been distributed into the test vessels) and in each test vessel at the end of the test. The range of the pH was 7.69 – 9.86 during the experiment. The pH of the control medium was not increased by more than 1.5 units during the test except one replicate. In this case the pH increased by 1.71 units, however this slight deviation was considered to be within the acceptable biological variability range and does not affect the results of the test.

Salinity:

Reconstituted water (ISO medium, according to OECD 202) was used as dilution water in the experiment. Separate stock solutions of individual substances are first prepared in deionised water (prepared in TOXI-COOP ZRT. by MILLIPORE ELIX 3 water purification system). The ISO medium is prepared by adding 25 mL from each of four stock solutions to one litre deionised water.

Nominal and measured concentrations:

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 first prepared by dispersing/dissolving an amount of 0.0804 g test item in 804 mL dilution water (OECDMedium). This solution was agitated by orbital shaker (~300 rpm) for a period of at least 2 days (approx. 3 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. Untreated control ran parallel in the test. After the formulation procedure algal cells were immediately introduced into test solution (start of experiment).

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

equivalent to 100 mg/L nominal

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 1.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

>= 1.04 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Details on results:

he growth inhibition calculated for the 72 hours exposure in treatment group compared to the control was 0.0 % for the average specific growth rate and 0.3 % for the yield. Based on the results of the statistical evaluation (T- Test; 2-tailed; α = 0.05) neither the 72-h average specific growth rate nor the 72-h yield showed significant difference compared to the control. Accordingly, the 72-h NOEC based on growth rate and yield was determined as the saturation concentration (equivalent to 100 mg/L nominal concentration; 1.04 mg/Lmeasured). The 72-h LOEC and EC50 values were determined to be higher than saturation(equivalent to > 100 mg/L nominal concentration; > 1.04 mg/L measured).

Validity criteria fulfilled:

yes

Conclusions:

In this 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item LEVOTHYROXINE ACID had no toxic effect at aquatic saturation (measured value: 1.04 mg/L; equivalent to 100 mg/L nominal concentration). Accordingly, the 72-h EC50 value was determined to be > 1.04 mg/L (equivalent to > 100 mg/L nominal concentration). The 72-h NOEC was determined to be 1.04 mg/L (equivalent to 100 mg/L nominal concentration). All validity criteria were met.

Executive summary:

Test item:LEVOTHYROXINE ACID(Batch No.: B486066)Test species:Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata); Strain number: 61.81 SAGExponentially-growing cultures; pre-culture was incubated for threedays before the test.Dilution water:OECD 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 1.04 mg/L (calculated as the geometric mean of measured start and end concentrations).Test design: Exponentially-growing cultures of Raphidocelis subcapitatawere exposed to the test item under defined conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included six replicates in the treatment group and for the untreated control. The alga cell concentration was approximately 104 cells/mL at the start of the test in all of the test cultures. Glass flasks with total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The alga cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, in 24-hour intervals. Analytics:Concentration of the test item were determined using HPLC- MSmethod at the start and at the end of the test.Endpoints:72-h EC10, EC20, EC50, NOEC and LOEC for both response variables (i.e. average specific growth rate and yield). Statistics:Statistical comparisons of average specific growth rates and yield in control and in treated group were carried out using Independent Samples T-Test (2-tailed; α= 0.05) by SPSS software.

Results:Validity:All validity criteria were met and therefore, the study can be considered as valid (see section 8.1)Analytical results:The mean measured test item concentration of the saturated test solution was 1.31 mg/L at the start and 0.83 mg/L at the end of the test. Biological results:The results of the statistical evaluation showed that the 0-72 h average specific growth rate and yield in the treatment group (saturated solution, equivalent to 100 mg/L nominal concentration)was not statistically significantly different from the untreated concurrent control. Accordingly, the 72-h NOEC was determined as the saturation concentration (equivalent to 100 mg/L nominal concentration; 1.04 mg/L measured).The 72-h EC50and LOEC were determined to be higher than saturation (equivalent to > 100 mg/L nominal concentration; > 1.04 mg/L measured).

Table 1: Summary of the Biological Endpoints

Endpoint	Growth rate (r)
72 h-EC10	> 1.04 mg/L (equivalent to > 100mg/L nominal)
72 h-EC20	> 1.04 mg/L (equivalent to > 100mg/L nominal)
72 h-EC50	> 1.04 mg/L (equivalent to > 100mg/L nominal)
72 h-NOEC	1.04 mg/L (equivalent to > 100mg/L nominal)
72 h-LOEC	> 1.04 mg/L (equivalent to > 100mg/L nominal)

 

Description of key information

In the 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item LEVOTHYROXINE ACID had no toxic effect at aquatic saturation (measured value: 1.04 mg/L; equivalent to 100 mg/L nominal concentration). Accordingly, the 72-h EC50 value was determined to be > 1.04 mg/L (equivalent to > 100 mg/L nominal concentration). The 72-h NOEC was determined to be 1.04 mg/L (equivalent to 100 mg/L nominal concentration). All validity criteria were met.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.04 mg/L