Formamide, N,N'-1,4-phenylenebis-, reaction products with 4-methyl-1,3-benzenediamine and sulfur, leuco derivatives

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:

2019-05-14 to 2019-05-17

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:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals, Series On Testing And Assessment OECD No.23 (Second Edition)

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L
- Sampling method: For determination of the test item concentrations six replicate samples (25 mL per replicate) from the test concentration and six replicate samples (25 mL per replicate) from the control group were taken at the start and at the end of the test. The samples taken were sent to individual analysis. The analysis was performed by the analytical laboratory of TOXI-COOP ZRT. according to the results of the analytical method validation (Study number: 805-100-2589 and 805-100-4227) by UV/VIS spectrophotometric method.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
The test item is poorly water soluble in the test medium. In order to achieve the maximum solubility level, the WAF method (according to OECD Series on Testing and Assessment No. 23) was applied. The test solution was prepared as follows: a supersaturated solution (100 mg/L nominal loading) was prepared by dispersing 0.1005 g of the test item into 1005 mL of the test medium and handled in ultrasonic bath for 10 minutes. This solution was mixed thoroughly for a period of 24 hours to achieve an equilibrated concentration and then filtrated through a membrane filter (0.45 μm) to separate the possible non-dissolved test material (corresponding to 100 mg/L nominal concentration). After the formulation procedure algal cells were immediately introduced into test solution (start of the experiment).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Breeding Conditions: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardized conditions according to the test guidelines.
- Method of cultivation: The pre-culture was prepared with OECD Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. (The pre-culture was incubated for four days before this test) The algal cultures used in this study did not contain deformed or abnormal cells.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

22.5 - 22.8°C (measured in the flask)
21.6 – 23.5°C (measured within the climate chamber)

pH:

7.60 – 9.09

Nominal and measured concentrations:

Nominal: 100 mg/L
Mean measured: 0.126 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks filled with volumes of 100 mL algal suspension per replicate
- Type: Closed. The test flasks were covered with air-permeable stoppers.
- Initial cells density: 10^4 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to guideline

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Light intensity and quality: The average light intensity measured at the position occupied by algal culture flasks was 6576 lux (SD: 78 lux), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: The cell concentration was determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
- Other: The cell morphology was examined and recorded at 24, 48 and 72 hours.

RANGE FINDING STUDY
In order to select appropriate test concentrations for use in the definitive test, a non-GLP preliminary range-finding test was conducted to determine the approximate toxicity of the test item. Due to the test item’s low solubility, preparation of test solution was performed using the WAF method (according to OECD Series on Testing and Assessment No. 23) as follows:
For preparation of the test solution, a supersaturated solution (100 mg/L nominal loading) was prepared by dispersing/dissolving an amount of 0.0404 g test item in 404 mL OECD medium and handled in ultrasonic bath for 10 minutes. This solution was mixed for a period of 24 hours to achieve an equilibrated concentration and then filtrated through a membrane filter (0.45 μm) to separate the possible non-dissolved test material. Untreated control ran parallel in the test.
Algal cells were exposed to the treatment group plus a control, for 72 hours. The test was performed with three replicates in the treatment group and with six replicates in the control.
The concentration levels used and results (72 h) of the preliminary range-finding test are summarised in table 1 in the section "Any other information on results including tables".
Based on the results of the non-GLP Preliminary Range-Finding Test, the main test was a limit test performed using the WAF method (according to OECD Series on Testing and Assessment No. 23) including a loading rate of 100 mg/L and a concurrent control group.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.126 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Growth rate and Yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

other: Growth rate and Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.126 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Growth rate and Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

other: Growth rate and Yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities: No abnormal shape of algal cells was noticed in the control and at the test item concentration of 0.126 mg/L (nominal 100 mg/L) during the study.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 72h ErC50: 0.83 mg/L, (95 % confidence limits: could not be calculated)
- 72h EyC50: 0.57 mg/L, (95 % confidence limits: 0.45 – 0.72 mg/L)

Reported statistics and error estimates:

Mean values and standard deviations were calculated for each treatment at the start and on each subsequent day of the test using Microsoft Excel for Windows software.
It was not necessary to analyse the average specific growth rate and yield in control and in treated group by statistical procedure, because there was only slight growth and yield increase and no inhibition. Therefore, LOEC and NOEC values were given directly from the raw data.

Table 1: Results of the Preliminary Range-finding Test

Test Group	Untreated control	WAF* (100 mg/L nominal)
Average of cell number at 72 hours (x 104cells/mL)	69.9	75.1
Growth Rates (µ) [0-72h] % Inhibition of µ [0-72h]	0.0589 -	0.0600 -1.9**
Yield (Y) [0-72h] % Inhibition of Y [0-72h]	68.9 -	74.1 -7.6**

* Water accomodated fraction

** Negative value means growth increase

Validity of the Test
- The cell density in the control cultures increased by a factor of 58.00 within 72 hours. This corresponds to a specific growth rate of 1.35 per day.
- The mean coefficient of variation (CV) for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72 h-tests) in the control cultures did not exceed 35 %
      - CV for section-by-section growth rate day 0-1: 24.79 %
      - CV for section-by-section growth rate day 1-2: 14.08 %
      - CV for section-by-section growth rate day 2-3: 5.28 %
The mean coefficient of variation for section-by-section specific growth rates: 14.72 %.
- The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 3.57 %.
All validity criteria were met, therefore the study can be considered as valid.

Analytical Results

One test concentration at saturation (water accommodated fraction, equivalent to 100 mg/L nominal concentration) and a concurrent control was tested in the main experiment. The concentration of the test item was analytically determined at the start and at the end of the test.
The measured concentrations were not within ± 20 % of the nominal during the experiment. Therefore the biological results are based on the calculated concentration based on the mean measured concentration and given as 0.126 mg/L according to Guideline OECD No. 23. No test item was detected in the control. Calculation of the test concentration is given in the following table.

 

Table 2: Calculation of the Test Concentration 

Nominal concentration (mg/L)	Mean measured concentration (mg/L)
	Start of the test (n=2)	End of the test (n=2)
100 (WAF*)	0.18**	0.13**
	0.21	0.18**
	0.20	0.18**
	0.18**	0.20
	0.13**	0.18**
	0.12**	0.13**
	0.135	0.117
Mean	0.126

  *WAF = water accommodated fraction (OECD No. 23.); loading rate: 100 mg/L nominal
**LOQ = limit of quantification (0.2 μg/mL), test concentration calculated as LOQ/2 (OECD No. 23.)

Validity criteria fulfilled:

yes

Conclusions:

In this 72-hour growth inhibition study on algae (Raphidocelis subcapitata), the obtained results showed that the test item had not any significant toxic effects on the test system.
The 72-h NOEC was determined to be 0.126 mg/L (nominal 100 mg/L).
The 72-h LOEC was determined to be > 0.126 mg/L (nominal > 100 mg/L).
All validity criteria were met. The results are based on mean measured test item concentrations.

Executive summary:

The effect of the test item Leuco Sulfur Brown 26 on the growth of a unicellular green algal species was determined in a growth inhibition study according to OECD guideline 201 and EU Method C.3.
Exponentially growing cultures of Raphidocelis subcapitata were exposed to the nominal concentration of 100 mg/L of the test item over a period of 72 hours in a limit test. Because of low solubility of the test item, test solution was prepared by utilizing the water accommodated fraction (WAF) approach. For determination of the test item concentration, samples were taken from the test concentration and control at the start and end of the test. Concentrations were determined using UV/VIS spectrophotometric method. Measured test item concentration did not remain within ± 20 % of the nominal over the test period of 72 hours, therefore the biological results are based on the mean measured test concentration.

The results obtained in this study showed that the test item Leuco Sulfur Brown 26 had not any significant toxic effects on the test system. The 72-h NOEC was determined to be 0.126 mg/L (nominal 100 mg/L). The 72-h LOEC was determined to be > 0.126 mg/L (nominal > 100 mg/L). All validity criteria were met.

Description of key information

In this 72-hour growth inhibition study on algae (Raphidocelis subcapitata), the obtained results showed that the test item had not any significant toxic effects on the test system.

The 72-h NOEC was determined to be 0.126 mg/L (nominal 100 mg/L).

The 72-h LOEC was determined to be > 0.126 mg/L (nominal > 100 mg/L).

All validity criteria were met. The results are based on mean measured test item concentrations.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.126 mg/L

Additional information

The effect of the test item Leuco Sulfur Brown 26 on the growth of a unicellular green algal species was determined in a growth inhibition study according to OECD guideline 201 and EU Method C.3.
Exponentially growing cultures of Raphidocelis subcapitata were exposed to the nominal concentration of 100 mg/L of the test item over a period of 72 hours in a limit test. Because of low solubility of the test item, test solution was prepared by utilizing the water accommodated fraction (WAF) approach. For determination of the test item concentration, samples were taken from the test concentration and control at the start and end of the test. Concentrations were determined using UV/VIS spectrophotometric method. Measured test item concentration did not remain within ± 20 % of the nominal over the test period of 72 hours, therefore the biological results are based on the mean measured test concentration.

The results obtained in this study showed that the test item Leuco Sulfur Brown 26 had not any significant toxic effects on the test system. The 72-h NOEC was determined to be 0.126 mg/L (nominal 100 mg/L). The 72-h LOEC was determined to be > 0.126 mg/L (nominal > 100 mg/L). All validity criteria were met.