N,N''-(methylenedi-4,1-phenylene)bis[N'-octyl]urea

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:

10 - 17 July 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The test was conducted according to OECD Guideline 201 and in compliance with GLP. A water accomodated fraction (WAF) was prepared by stirring for about 48 hours and subsequent filtration. The measured concentration of test substance was below the limit of quantification (1.0 mg/L) at both measurement times (start and end of the study). Therefore, the exposure has not been confirmed by analytical measurements. No effects were observed up to a loading rate of 100 mg/L. Based on this result it can be concluded that the EC50 value is higher than the maximum solubility in medium. Therefore the endpoint algae toxicity is assumed to be sufficiently covered by this study and a reliability 2 is assigned

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

(2006, Annex 5 corrected, July 28, 2011)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document No. 23 Guidance document on testing of difficult substances and mixtures (2000)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples were taken from both control treatment and the WAF prepared at a loading rate of 100 mg/L
- Sampling method: At the start of the test, a separate vessel that was prepared especially for sampling purposes was sampled. At the end of the test, samples were taken from a pooled solution (equal volume of each test vessel).
- Sample volume: Appr. 100 - 120 mL
- Sample storage conditions before analysis: Not reported

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A Water Accomodated Fraction (WAF) was prepared at a loading rate of 100 mg/L by adding the test substance to medium, stirring for 48 h and subsequent filtration with a glass fiber filter (GB-140, 0.4 µm pore size)
- Evidence of undissolved material (e.g. precipitate, surface film, etc): At the start of the exposure, the test solutions were colorlous and clear. At the end of the exposure they were green, due to algae growth. There are no visual observations reported of the test dispersion prior to filtration.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: ATCC 22622
- Source: American Type Culture Collection
- Method of cultivation: under sterile conditions

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: not reported

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Not reported

Test temperature:

22.2 - 22.6 °C

pH:

7.8 - 7.9

Nominal and measured concentrations:

Nominal:
Limit: Loading rate 100 mg/L
Measured:
The measured concentrations were below the LOQ (< 1.0 mg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterilised 500 mL Erlenmeyer flask (with gas-permeable silicon rubber plug)
- Fill volume: 100 mL
- Aeration: not aerated, but constantly agitated (vessels were shaken at appr. 100 rpm)
- Initial cells density: 0.75 x 10^4 cells/mL
- Control end cells density: 99 x 10^4 cells/mL
- No. of vessels containing the WAF prepared at a loading rate of 100 mg/L (replicates): 6
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified water
- Culture medium different from test medium: no data


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 88 - 90 µmol/m2/s, 400 - 700 nm


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Cell concentrations were determined every 24h after the start of the exposure with a Coulter counter.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (June 2015)

Duration:

72 h

Dose descriptor:

EC50

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: The EC50 was > maximum soluble concentration of test substance in medium.

Duration:

72 h

Dose descriptor:

NOEC

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: The NOEC = maximum soluble concentration of test substance in medium.

Results with reference substance (positive control):

72h-ErC50: 1.3 mg/L
This value was within the normal range of the reference substance at the test laboratory (mean ± 2 SD): Mean 72h EC50 ± SD: 0.98 ± 0.19 mg/L (n = 27)

Reported statistics and error estimates:

After an F-test was done to determine the homogeneity of variance for the data, a Student's t-test was used to estimate the significant difference compared with the control. The statistical analysis was carried out with MS Excel.

No significant inhibition of growth was observed in the WAF prepared at a loading rate of 100 mg/L.

Validity criteria fulfilled:

no

Remarks:

See 'Remarks on results including tables and figures'

Conclusions:

Since no significant growth inhibition was observed in the WAF prepared at a loading rate of 100 mg/L, it is concluded that the 72h-EC50 to algae is > the maximum soluble concentration of the test substance in medium and the NOEC is arbitrarily set at the maximum soluble concentration of the test substance in medium.

Executive summary:

The toxicity of KY-EU to Pseudokirneriella subcapitata was examined according to OECD guideline 201 and GLP.

Algae were exposed to a WAF that was prepared at a loading rate of 100 mg/L by stirring for 48h, followed by filtration through a a filter with 0.4 µm porosity. Samples that were taken at the beginning and the end of the test were analysed with HPLC-UV. The analysis was based on total peak area of two detected peaks.

There is no evidence of exposure to the main components of KY-EU in these tests. The measured concentrations of test substance were below the limit of quantification (1.0 mg/L) at all measurement times.

No significant growth inhibition was observed in the WAF prepared at a loading rate of 100 mg/L. Based on this result it can be concluded that the 72h-EC50 to algae is > the maximum soluble concentration of the test substance in medium and the NOEC is arbitrarily set at the maximum soluble concentration of the test substance in medium.

Therefore the endpoint algae toxicity is assumed to be sufficiently covered by this study and a reliability 2 is assigned.

Description of key information

The 72h-EC50 to algae is > the maximum soluble concentration of the test substance in medium.
The NOEC is arbitrarily set at the maximum soluble concentration of the test substance in medium.

Additional information

There are two studies available. Only the study from Ono (2015) is regarded as reliable (with restrictions) and therefore this study has been selected as key study.

In the study by Ono (2015), which was carried out according to OECD201 and GLP, algae were exposed to a WAF that was prepared at a loading rate of 100 mg/L by stirring for 48h, followed by filtration through a a filter with 0.4 µm porosity. Samples that were taken at the beginning and the end of the test were analysed with HPLC-UV. The analysis was based on total peak area of two detected peaks.

There is no evidence of exposure to the main components of KY-EU in these tests. The measured concentrations of test substance were below the limit of quantification (1.0 mg/L) at all measurement times.

No significant growth inhibition was observed in the WAF prepared at a loading rate of 100 mg/L. Based on this result it can be concluded that the 72h-EC50 to algae is > the maximum soluble concentration of the test substance in medium and the NOEC is arbitrarily set at the maximum soluble concentration of the test substance in medium.

Therefore the endpoint algae toxicity is assumed to be sufficiently covered by this study and a reliability 2 is assigned

In the study by L'Haridon (2003) which was carried out according to OECD 201 and GLP, algae were exposed to WAFs that were prepared at loading rates of of 62.5, 125, 250, 500, 1000, 2000 and 4000 mg/L by stirring for 22h, followed by filtration through a filter with 4-7 µm porosity, and a filter with 0.45 µm porosity, subsequently. Samples that were taken at the beginning and the end of the test and stored at -20°C were analysed with HPLC-UV. The analysis was based on only one component out of 3 of KY-EU.

Although the report states effects of the test item to algae, there is no evidence of exposure to the main components of KY-EU in these tests. The measured concentrations of test substance were below the limit of quantification (0.5 mg/L) at all measurement times. No effects were observed up to a loading rate of 1000 mg/L. Based on this result it is normally concluded that the EC50 value is higher than the maximum solubility in medium. However, for unknown reasons, higher loading rates were tested in this study (2000 and 4000 mg/L). At these loading rates, toxicity was observed (>50% at the highest loading) following a dose-response relationship. This indicates that exposure to an unknown component has taken place and considering the dose-relationship, this component had not yet reached its solubility level (e.g. an impurity in KY-EU). Since the component causing the toxicity is not known and the concentration of this component is not known, a meaningful EC50 value cannot be derived. Therefore the study is considered to be not reliable and disregarded.