Phenol, 4-(phenylamino)-, sulfurized, leuco deriv.

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 May 2012 to 01 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 761/2009 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), 2009, C.26: Lemna

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

swissmedic, decision: 19-November-2010

Analytical monitoring:

no

Details on sampling:

On request of the Sponsor no analytical work was performed.

Details on test solutions:

Due to the low water solubility of the test item, a dispersion with the loading rate of 100 mg/L was prepared at the start of the test and before each test medium renewal by dispersing 100 mg (100.02 to 100.06 mg) of the test item in 1000 mL of test water. This preparation was supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer over 96 hours at room temperature in the dark to dissolve a maximum amount of the test item in the dispersion. No auxiliary solvent or emulsifier was used.

After the 96-hour stirring period, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC20, pore size 0.2 µm).

The undiluted filtrate was used as test medium. The test medium was prepared just before the start of the exposure and before each test medium renewal. The preparation of the test medium was performed as far as possible in the dark to avoid photolytic degradation of the test item.

The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

Test organisms (species):

Lemna gibba

Details on test organisms:

The test organism used for the study was the duckweed Lemna gibba G3 (family Lemnaceae, Macrophyta). Lemna gibba is a preferred aquatic macrophyte species used to evaluate the toxicity of test items to higher aquatic plants.

The original culture was supplied by Bayer CropScience AG, 40789 Monheim, Germany in 2007. The plants were axenically cultivated in the laboratories of Harlan Laboratories Ltd. for more than four weeks prior to the test and under standardized conditions in the same nutrient medium as used in the test.

The pre-culture was maintained under the conditions of the test (nutrient medium, light conditions and temperature) for seven days prior to the start of the test. The test was started with plants from an exponentially growing culture. Only young, rapidly growing colonies without visible lesions were used.
The test method and the test species are recommended by the test guideline.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

7 d

Hardness:

3.0 mmol/L (= 300 mg/L as CaCO3)

Test temperature:

The water temperature was 24-25°C during the test period (see attached Table 9).

pH:

At the start of the test medium renewal periods, the pH of the test medium and the control ranged from 7.5 to 8.2 (see attached Table 8). At the end of the renewal periods, pH values between 8.3 and 9.4 were measured.

Dissolved oxygen:

not applicable

Salinity:

according to OECD test guideline

Nominal and measured concentrations:

A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the plants up to its limit of solubility in the test water. The undiluted filtrate with the loading rate of 100 mg/L of C.I. Leuco Sulphur Brown 96 was tested. Additionally, a control was run in parallel (test water without addition of the test item).

The limit test was based on the results of a range-finding test and on results of a pre-experiment to determine the solubility of the test item.

Loading rates exceeding 100 mg/L were not tested in accordance with the test guidelines.

Details on test conditions:

The test vessels were incubated in a temperature-controlled water bath with a temperature of 24-25 °C. They were continuously illuminated using fluorescent tubes (Philips TLD 36W 1/840) installed above the test vessels in order to achieve a light intensity of 7120–8180 Lux.

A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the plants up to its limit of solubility in the test water. The undiluted filtrate with the loading rate of 100 mg/L of C.I. Leuco Sulphur Brown 96 was tested. Additionally, a control was run in parallel (test water without addition of the test item).

The limit test was based on the results of a range-finding test and on results of a pre-experiment to determine the solubility of the test item.

Loading rates exceeding 100 mg/L were not tested in accordance with the test guidelines.

The test design included six replicates of the single test concentration and six replicates of the control.

The plants were exposed to the test item for a period of seven days in a semi static test with test medium renewal every 48 or 72 hours. The semi-static test design was chosen to keep the concentrations of the test item and nutrients in the test media as constant as possible during the exposure period.

At the start of the test, Lemna colonies were transferred aseptically from the pre-culture into the different test vessels in a randomized order. The test was started with three randomly selected colonies per vessel. At the test medium renewal dates, the test plants were transferred under aseptic conditions to clean test vessels with freshly prepared test medium of the corresponding concentration.

The test duration was 7 days.

Due to the low water solubility of the test item, a dispersion with the loading rate of 100 mg/L was prepared at the start of the test and before each test medium renewal by dispersing 100 mg (100.02 to 100.06 mg) of the test item in 1000 mL of test water. This preparation was supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer over 96 hours at room temperature in the dark to dissolve a maximum amount of the test item in the dispersion. No auxiliary solvent or emulsifier was used.

After the 96-hour stirring period, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC20, pore size 0.2 µm).

The undiluted filtrate was used as test medium. The test medium was prepared just before the start of the exposure and before each test medium renewal. The preparation of the test medium was performed as far as possible in the dark to avoid photolytic degradation of the test item.

Reference substance (positive control):

yes

Remarks:

testet twice a year

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

growth rate

Details on results:

Biological Results
The calculations are based on the theoretical concentration as no analytical determination could be conducted.

The influence of the test item C.I. Leuco Sulphur Brown 96 on the growth of Lemna gibba is shown in the attached Table 1 to Table 6 and Figure 1. The section-by-section growth rates (calculated on the basis of frond numbers) are given in the attached Table 7.

The test item C.I. Leuco Sulphur Brown 96 had no statistically significant inhibitory effect on the growth of Lemna gibba (growth rate and yield based on frond number and dry weight) after the exposure period of 7 days at the theoretical concentration of 100 mg/L (undiluted filtrate) (results of Student t-tests, one-sided smaller, alpha = 0.05, see attached Table 3 to Table 6).

No abnormalities in appearance of the test plants were recorded.


Validity Criteria
The doubling time (Td) of frond number in the control was calculated to be 1.7 days (Td = ln 2 / µ). According to the test guideline, the validity criterion for the study (Td < 2.5 days corresponding to an average growth rate of 0.275 day-1) was fulfilled, indicating that the Lemna growth was satisfactory under the test conditions.

The test medium was pink colored and the test item was uniformly distributed in the test water during the entire test duration.

Before test start, the average light intensity was 7600 Lux, spanning a range of 7120 to 8180 Lux. The light intensity over the test area did not exceed the range of ±15% of the mean value.

At the start of the test medium renewal periods, the pH of the test medium and the control ranged from 7.5 to 8.2 (see attached Table 8). At the end of the renewal periods, pH values between 8.3 and 9.4 were measured. The increase of the pH during the renewal periods was caused by the CO2 consumption of the plants due to their growth. The water temperature was 24-25°C during the test period (see attached Table 9).

Results with reference substance (positive control):

For evaluation of the sensitivity of the test system, the reference item 3,5-dichlorophenol is tested twice a year. The result of the latest positive control test performed in October 2012 showed that the sensitivity of the test system was within the historical range (7-day EC50 for the growth rate based on frond numbers: 10 mg/L, study D64322; internal historical range from 2003 to 2012: 8.0-11 mg/L).

Reported statistics and error estimates:

The EC10, EC20 and EC50 values for the inhibition of the growth rate and yield based on frond numbers and dry weight and their 95% confidence limits could not be determined because of the absence of a significant inhibitory effect of the test item on the algal growth at the tested concentration. They were directly determined from the raw data.

The NOEC and the LOEC for the different growth parameters were determined by testing the parameters at the test concentration for statistically significant differences to the control values using Student t-test .

For further information (Tables) please see the attachments.

Validity criteria fulfilled:

yes

Conclusions:

The test item C.I. Leuco Sulphur Brown 96 had no toxic effects on Lemna gibba during the 7-day test period up to its solubility limit in the test water under the present test conditions.

Executive summary:

The influence of the test item C.I. Leuco Sulphur Brown 96 on the growth of the freshwater aquatic plant Lemna gibba (duckweed) was investigated in a 7‑day semi‑static test, based on the OECD Guideline No. 221 “Lemnasp. Growth Inhibition Test” (2006) and the Commission Regulation (EC) No. 761/2009, C.26.

 

Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was ultrasonic treated for 15 minutes and continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered. The undiluted filtrate with the theoretical concentration of 100 mg/L was used as test medium. Additionally, a control was tested in parallel.

 

The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (2000).

 

No analytical work was performed on request of the Sponsor due to the specific properties of the test item (low solubility in water).

 

In conclusion, the test item C.I. Leuco Sulphur Brown 96 had no toxic effects on Lemna gibba during the 7-day test periodup to its solubility limit in the test water under the present test conditions.

 

Description of key information

The test item C.I. Leuco Sulphur Brown 96 had no toxic effects on Lemna gibba during the 7-day test period up to its solubility limit in the test water under the present test conditions.

7d-EC50 (growth rate) > 100 mg/L

7d-LOEC (growth rate) > 100 mg/L

7d-NOEC (growth rate) > 100 mg/L

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:

100 mg/L

Additional information

The influence of the test item C.I. Leuco Sulphur Brown 96 on the growth of the freshwater aquatic plant Lemna gibba (duckweed) was investigated in a 7‑day semi‑static test, based on the OECD Guideline No. 221 “Lemnasp. Growth Inhibition Test” (2006) and the Commission Regulation (EC) No. 761/2009, C.26.

Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was ultrasonic treated for 15 minutes and continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered. The undiluted filtrate with the theoretical concentration of 100 mg/L was used as test medium. Additionally, a control was tested in parallel.

The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (2000). No analytical work was performed on request of the Sponsor due to the specific properties of the test item (low solubility in water).

In conclusion, the test item C.I. Leuco Sulphur Brown 96 had no toxic effects on Lemna gibba during the 7-day test period up to its solubility limit in the test water under the present test conditions.