2[2'-Hydroxy-5'-(phenyl)ureylenphenyl]benzothiazole

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Mar 2018 - 06 Apr 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011 and • Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, 2000

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

no

Remarks:

No analytical monitoring was carried out due to the low solubility of the test item .

Details on sampling:

- Concentrations:
Solutions containing 1.0, 10 and 100% of the SS prepared at a loading rate of 100 mg/L.
3 replicates of each test concentration,
6 replicates of the control and limit concentration,
1 extra replicate of each test group for sampling purposes after 24 hours of exposure,
1 or 2 replicates of each test concentration without algae
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

- Sampling method:
Samples for possible analysis were taken from all test concentrations and the control.
In addition, the filter containing the undissolved residue was kept for possible analysis.
Frequency at t=0 h, t=24 h and t=72 h
Volume 2.0 mL from the approximate centre of the test vessels.

- Sample storage conditions before analysis:
Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Test solutions

Vehicle:

no

Details on test solutions:

The substance tested was a slight yellow powder with a purity of 98% which was not completely soluble in test medium at the loading rate initially prepared.
No correction was made for the purity/composition of the test item.
Preparation of test solutions started with a loading rate of 100 mg/L applying an overnight period of magnetic stirring to ensure maximum dissolution of the test item in medium.
Thereafter, the aqueous Saturated Solution (SS) was collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.
Any residual volumes were discarded.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)
- Strain: strain: NIVA CHL 1
- Source: In-house laboratory culture.
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10E4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

21-24°C.

pH:

8.1 -8.3

Nominal and measured concentrations:

Solutions containing 1.0, 10 and 100% of the SS prepared at a loading rate of 100 mg/L.
Samples taken from the undiluted SS were analysed. Test item concentrations were below the Limit of Quantification (LOQ), i.e. 0.008 mg/L, throughout the test. Nevertheless, it can be stated that testing was ultimately performed at the maximum soluble concentration of test item in test medium. Analysis of the filter residue demonstrated the use of the test item during preparation of test solutions.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Static
- Aeration: continuous
- Initial cells density: An initial cell density of 1 x 10E4 cells/mL.
- Control end cells density: In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 130).
- No. of organisms per vessel: 1 x 10E4 cells/mL.
- No. of vessels per concentration (replicates): 3 x 104 cells/mL for each test concentration / 6 x 10E4 cells/mL for limit concentration
- No. of vessels per control (replicates): 6 x 10E4 cells/mL.

GROWTH MEDIUM
- Standard medium used: yes
Stock culture medium
M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)

NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

TEST MEDIUM / WATER PARAMETERS
Pre-culture medium M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2

- Source/preparation of dilution water: MMilli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 24h (Continuously using TLD-lamps with a light intensity within the range of 68 to 73 µE.m-2.s-1.)
- Light intensity and quality: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm
- Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

EFFECT PARAMETERS MEASURED
- pH recorded at the beginning and the end of the test
- The temperature measured in the incubator was maintained between 21 and 22°C.
- Appearance of the cells:
At the end of the final test microscopic observations were performed on the limit concentration to observe for any abnormal appearance of the algae compared to the control.
- Determination of cell concentrations:
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length =10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.

TEST CONCENTRATIONS
Test item Solutions containing 1.0, 10 and 100% of the SS prepared at a loading rate of 100 mg/L.
Control Test medium without test item or other additives.
Replicates 3 replicates of each test concentration,
6 replicates of the control and limit concentration,
1 extra replicate of each test group for sampling purposes after 24 hours of exposure,
1 or 2 replicates of each test concentration without algae.

A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest test concentration. Lower concentrations were prepared by diluting the highest test concentration in test medium.
A combined limit/range-finding test was performed. Six exponentially growing algal cultures per group were exposed to an untreated control and to an undiluted SS prepared at a loading rate of 100 mg/L in the limit test. In addition, three replicates per group were exposed to solutions containing 1.0 and 10% of the SS in a combined range-finding test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

No significant differences were recorded between the values for growth rate or yield at any of the test concentrations when compared to the control group.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the limit concentration when compared to the control.

Results with reference substance (positive control):

- Results with reference substance valid? yes

The EC50 for growth rate inhibition (72h-ERC50) was 1.6 mg/L with a 95% confidence interval ranging from 1.5 to 1.6 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.

The EC50 for yield inhibition (72h-EYC50) was 0.50 mg/L with a 95% confidence interval ranging from 0.48 to 0.52 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L. Hence, the 72h-EYC50 for the algal culture tested corresponds with this range.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), no inhibition of growth rate and yield was recorded at any of the concentrations of the tested substance.

The EL50 for growth rate (72h-ERL50) and yield (72h-EYL50) inhibition was beyond the range of concentrations tested, i.e. exceeded a loading rate of 100 mg/L.
The 72h-NOEL for growth rate inhibition and yield inhibition was at 100 mg/L.

Executive summary:

The objective of the study was to evaluate test item for its ability to generate toxic effects inRaphidocelis subcapitata(formerly known as Pseudokirchneriella subcapitata)during an exposure period of 72 hours and, if possible, to determine the NOEL, EL₁₀and EL₅₀for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

The batch of test item tested was a slight yellow powder with a purity of 98% which was not completely soluble in test medium at the loading rate initially prepared.A SaturatedSolution (SS) was prepared at a loading rate of 100 mg/L and used as the highest test concentration. Lower concentrations were prepared by diluting the highest test concentration in test medium.

A combined limit/range-finding test was performed. Six exponentially growing algal cultures per group were exposed to an untreated control and to an undiluted SS prepared at a loading rate of 100 mg/L in the limit test. In addition, three replicates per group were exposed to solutions containing 1.0 and 10% of the SS in a combined range-finding test. The initial algal cell density was 1 x 10⁴cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

No significant differences were recorded between the values for growth rate or yield at any of the test concentrations when compared to the control group.

Samples taken from the undiluted SS were analysed. Test item concentrations were below the Limit of Quantification (LOQ), i.e. 0.008 mg/L, throughout the test. Nevertheless, it can be stated that testing was ultimately performed at the maximum soluble concentration of test item in test medium. Analysis of the filter residue demonstrated the use of the test item during preparation of test solutions.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study are summarized in the table below.

Parameter (mg/L)	NOEL	EL10	EL50
Growth rate	100	>100	>100
Yield	100	>100	>100

Due to the very low solubility of test item in water, concentration levels that might be toxic for algae could not be reached.