N,N’-di[3-(p-toluene sulfonyl)oxy]phenyl 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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

March 23 2006. Annex 5 corrected, July 28, 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: "Algal Growth Inhibition Test" - Testing method for New Chemical Substances of Japanese Chemical Substances Control Law

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document No. 23

Version / remarks:

February 2019
Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: single concentration (nominal concentration: 100 mg/L)
- Sampling method: Solution sampled separately from the preparation container (at start of exposure); mixed solution was taken out with equal volumes of the test solution in the test vessels in each test level (at the end of exposure). Algae removed by centrifugation (3000rpm, 10 minutes)
- Sample storage conditions before analysis: samples were analysed straightaway after collection.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution was prepared. 100g test item and 1000mL medium were mixed to produce the test concentration 100 mg/L and stirred by a magnetic stirrer for 48h to prepare the test solution. The test solution was divided into each test vessel.
- Controls: Culture medium (negative control) prepared using the same method as the test solution but no test item added.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): No used; not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): not applicable
- Test concentration separation factor: not applicable (limit test)
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): yes: suspension, precipitation and insoluble matter were observed.
- Other relevant information: undissolved test item had no physical effect on the test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Algae
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection
- Method of cultivation: Passage cultured under sterile conditions in test facility

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

As recommended in guideline.

Post exposure observation period:

Not applicable

Test temperature:

22.3 - 22.8°C

pH:

7.7 - 8.1

Nominal and measured concentrations:

Nominal: 100 mg/L
Measured: 0.00102 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flask with gas-permeable Silicosen
- Material, size, headspace, fill volume: Glass, 300 mL, yes, 100mL/vessel
- Aeration: continuous shaking
- Initial cells density: 0.75 x 10E4 cells/mL (CFV = 28)
- Control end cells density: CFV = 4500
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: Nominal 90µmol-m-2.s-1 in wavelength of 400-700nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Spectrophotofluorometer
- Chlorophyll measurement: yes biomass - (CFV)


TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable - limit test
- Justification for using less concentrations than requested by guideline: Not applicable

RANGE FINDING STUDY
- Test concentrations: 100 mg/L (saturated solution)
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Not conducted in parallel with study but lab conducts positive controls periodically to confirm the reproducibility of the test system. Period of study - March 26 - April 29, 2019 72h ErC50 = 1.1mg/L

Duration:

72 h

Dose descriptor:

EC50

Remarks:

growth rate

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks on result:

other: No growth rate inhibition was observed during the test.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.001 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Remarks on result:

other: No growth rate inhibition was observed in the study.

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: yes, precipitation was observed
- Effect concentrations exceeding solubility of substance in test medium: no effects observed.

Results with reference substance (positive control):

72h ErC50 = 1.1mg/L

Reported statistics and error estimates:

See Table 3

Table 1: Value of biomass at each time

Measured concentration (mg/L)	No.	Chlorophyll fluorescence value (relative unit)
		0 hours^a	24 hours	48 hours	72 hours
Control	A	28	210	1200	5100
	B	28	210	1400	5300
	C	28	220	1300	5200
	D	28	210	1200	4900
	E	28	210	1300	5100
	F	28	200	1300	4500^b
	Mean	28	210	1300	5000
	S.D.	0	5.7	57	280
0.00102	A	28	200	1200	5000
	B	28	200	1300	4900
	C	28	200	1300	5000
	D	28	190	1300	4900
	E	28	200	1200	5100
	F	28	200	1300	4900
	Mean	28	200	1300	5000
	S.D.	0	2.6	46	100

^{b: The value based on the measured value of pre-culture}

^{c: The minimum cell growth in the control (bioass at the end of exposure / biomass at the start of exposure) 4500/28 = 161}

Table 2: Growth rate and growth inhibition rate

Measured concentration (mg/L)	No.	Growth rate (0-3d)	Growth inhibition rate (%)
Control	A	1.74	-
	B	1.75	-
	C	1.74	-
	D	1.72	-
	E	1.73	-
	F	1.69	-
	Mean	1.73	-
	S.D.	0.0196	-
0.00102	A	1.73	-0.073
	B	1.72	0.28
	C	1.73	-0.24
	D	1.72	0.47
	E	1.74	-0.53
	F	1.72	0.40
	Mean	1.73	0.050
	S.D	0.00686	0.40

 

Table 3: Result of statistical analysis

Measured concentration (mg/L)	Statistical analysis	Statistical procedure
0.00102	n.a.	F test Aspin-Welch t test

Table 4: Variation of growth rate in control

<variation for section-by-section specific growth rates in the controls>

Control No.	Mean	Standard deviation	Coefficient of variation (%)
A	1/74	0.296	17	20 (mean)
B	1.75	0.364	21
C	1.74	0.355	20
D	1.72	0.334	19
E	1.73	0.322	19
F	1.69	0.378	22

<variation of average specific growth rates in replicate controls>

	0-3 days
Mean	1.73
Standard deviation	0.0196
Coefficient of variation (%)	1.1

Table 5: Measured concentration of the test item in test solution

Nominal concentration (mg/L)	Measured concentration (mg/L)  (Percentage of measured concentration versus that at the start %)
	At the start	At the end	Time weighted mean
Control	<LOQ	-	-
100	0.000113	0.00368	0.00102

LOQ: 0.0000501 mg/L

 

The time weighted mean is calculated by the following calculation:

[72 (C₀-C₇₂)/(lnC₀-lnC₇₂]/72

C_n: the measured concentration at n hours

lnC_n: the natural logarithm of C_n

 

Validity criteria fulfilled:

yes

Remarks:

-Cell growth in controls = Factor 161 (criteria: 16) - Mean coefficient of variation for section-by-section specific growth rates = 20% (criteria <35%) - Coefficient of variation of average specific growth rates in replicate controls = 1.1% (criteria <7%)

Conclusions:

No growth inhibition was observed during the limit test. The 72h EC50 was calculated as >0.00102 mg/L greater than the solubility of the test item in the test medium.

Description of key information

In a study conducted in accordance with OECD Guideline 201 on Pseudokirchneriella subcapitata, the 72h ErC50 >100 mg/L (nominal) was reported.  Based on measured concentrations, no mortality or abnormal behaviour was observed up to the limit of solubility of the test item (TWA measured with analytical verification at 0.00102 mg/L). 

Additional information

The 72h EC50 value is >100 mg/L it is not possible to report this as the key value for chemical safety assessment in the boxes above.