N,N''-(4-methyl-m-phenylene)bis[N',N'-dimethylurea]

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:

30 November 2011 - 20 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): TDI-Urone
- Substance type: organic
- Physical state: clear colourless powder
- Analytical purity: 97.5 % active content (being a mixture of the 4-methyl isomer and the 2-methyl isomer)
- Isomers composition: ratio of 4-methyl isomer and the 2-methyl isomer: approximately 80:20
- Lot/batch No.: 1268
- Expiration date of the lot/batch: 27 October 2012 (allocated by NOTOX, 1 year after receipt of the test substance)
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Sampling for analysis of test concentrations:
Singular samples for possible analysis were taken from all test concentrations and the control according to the schedule below. The method of analysis is described in the appended Analytical Report.
- Frequency at t=0 h, t=24 h and t=72 h
- Volume: 1.8 ml
- Storage: Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test
period.

Additionally, singular reserve samples of 1.8 ml were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

no

Details on test solutions:

Preparation of test solutions
The standard test procedures required generation of test solutions, which contained completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that would disturb the test system was prevented as much as possible (e.g.
film of the test substance on the water surface).

The batch of TDI-Urone tested was a clear colourless powder with a purity of 97.5% active content (being a mixture of isomers) and completely soluble in test medium at the concentrations tested.

Preparation of test solutions started with the highest test concentration of 100 mg/l applying a short period of magnetic stirring (~10 minutes) to ensure complete dissolution in test medium. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless.

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 10^4 cells/ml.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Species: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Reason for selection: This system is an unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.
- Age of inoculum (at test initiation):
- Method of cultivation:


FRESH WATER ALGAE CULTURE
- Stock culture: 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.
- Light intensity: 60 to 120 µE/m²/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
- Stock culture medium: see section "Any other information on materials and methods incl. tables".
- Pre-culture: 3 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 10^4 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Pre-culture medium: see section "Any other information on materials and methods incl. tables".

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

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Not applicable.

Hardness:

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

Test temperature:

Temperature of medium: 23.0 °C
Temperature range: 21.8 - 22.4 °C

pH:

7.9 - 8.0

Dissolved oxygen:

Not applicable.

Salinity:

Not applicable.

Nominal and measured concentrations:

Combined limit/range-finding test:
- nominal: 0.10, 1.0, 10 and 100 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml, all-glass, containing 50 ml of test solution
- Aeration: no
- Initial cells density: 10^4 cells/ml
- Control end cells density: Test medium without test substance or other additives.
-Replicates:
* 6 replicates of the control and the highest test concentration,
* 3 replicates of the lower test concentrations,
* 1 extra replicate of each test group for sampling purposes,
* 1 or 2 replicates of each test concentration without algae.
- Incubation: Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.


TEST MEDIUM / WATER PARAMETERS
- Medium: M2 (see "Any other information on materials and methods incl. tables" for details)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Light intensity and quality: Continuously using TLD-lamps of the type „Cool-white‟ of 30 Watt, with a light intensity within the range of 75 to 88 µE./(m² s)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- 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 720 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions.
- Other: Appearance of the cells: At the end of the final test microscopic observations were performed on the highest test concentration to observe for any abnormal appearance of the algae.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

No details reproted as no effects could be observed at the highest concentration tested (100 mg/L).

Results with reference substance (positive control):

Pseudokirchneriella subcapitata, strain: NIVA CHL-1. Fresh water algal growth inhibition test with potassium dichromate (NOTOX Project 498547).
- Start of first exposure: 02 January 2012
- Completion last exposure: 05 January 2012
The study procedures described in this report were based on the OECD guideline No. 201, Adopted March 23, 2006 and ISO Standard 8692, Second edition, 01 October 2004.
This reference test was carried out to check the sensitivity of the test system used by NOTOX to Potassium dichromate (Merck, Art. 1.04864, Batch K34869764 607).
Algae were exposed for a period of 72 hours to K 2 Cr 2 O 7 (Potassium dichromate) concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l and toa control. The initial cell density was 1.0 x 10^4 cells/ml.

Results:
Potassium dichromate reduced growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/l and higher.
The EC50 for growth rate reduction (ErC50: 0-72h) was 1.8 mg/l with a 95% confidence interval ranging from 1.4 to 2.4 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ErC50: 0-72h for the algal culture tested corresponds with this range.
The EC50 for yield inhibition (EyC50: 0-72h) was 0.69 mg/l with a 95% confidence interval ranging from 0.50 to 0.95 mg/l. The historical ranges of the 72h-EC50 for yield inhibition lie between 0.43 and 1.1 mg/l. Hence, the E Y C 50 : 0-72h for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

Not applicable.

1)           Measured test substance concentrations

The results of analysis of the samples taken during the combined limit/range-finding test are described in Table 2.

 

Analysis of the samples taken from 100 mg/l at the start, after 24 hours and at the end of the test showed that the measured concentration was stable and in agreement with nominal (101-105%)

 

Table 1: Procedural recovery samples

Date of preparation [dd-mm-yy]	Date of analysis [dd-mm-yy]	Target concentration [mg/l]	Nominal concentration [mg/l]	Analysed concentration [mg/l]	Recovery [%]	Mean recovery [%]
16-01-12	16-01-12	1	1.00	1.02	102	100
			1.00	0.992	99
16-01-12	16-01-12	100	100	101	100	100
			100	101	100

 

Table 2: Concentrations of the test substance in test medium 

Time of sampling [hours]	Date of preparation [dd-mm-yy]	Date of analysis [dd-mm-yy]	Concentration		Relative to nominal [%]	Relative to initial [%]
			Nominal [mg/L]	Analysed [mg/L]
0	09-01-12	16-01-12	0	n.d.	n.a.
			100	101	101
			100²	103	103
24	10-01-12	16-01-12	0	n.d.	n.a.	n.a.
			100	102	102	101
			100²	105	105	102
72	12-01-12	16-01-12	0	n.d.	n.a.	n.a.
			100	103	103	101
			100²	101	101	98

¹Samples were stored in the freezer (≤ -15°C) until the day of analysis.

²Without algae. 

n.d.     Not detected.

n.a.     Not applicable.

 

 

2)           Mean cell densities

Table 3 shows mean cell densities measured at 24-hour intervals at the different concentrations of TDI-Urone.

 

Table 3: Mean cell densities (x 10⁴cells/ml) 

Concentration TDI-Urone [mg/l]	Exposure time (hours)
	0	24	48	72
Control	1.0	8.5	39.7	164.4
0.10	1.0	8.9	40.2	163.6
1.0	1.0	8.4	37.1	155.0
10	1.0	8.6	36.5	146.1
100	1.0	8.6	38.7	154.1

 

 

3)   Reduction of growth rate and inhibition of yield

Table 4 shows the calculation of the percentages of growth rate reduction (total test period) and the percentages of yield inhibition. Table 5 shows the calculation of the percentages of growth rate reduction at different time intervals 

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 exposed cells when compared to the control. 

          

Table 4: Percentage reduction of growth rate (total test period) and percentage inhibition of yield 

Concentration TDI-Urone [mg/l]	Mean growth rate		Yield (0-72 h)
	µ (0-72 h)	Reduction (%)	x 10^4cells/ml	Inhibition (%)
Control	0.07082		163.41
0.10	0.07075	0.1	162.58	0.5
1.0	0.07004	1.1	154.01	5.7
10	0.06922	2.3	145.06	11.2
100	0.06989	1.3	153.09	6.3

 

 

Table 5: Percentage reduction of growth rate at different time intervals 

Concentration TDI-Urone [mg/l]	Mean growth rate
	µ (0-24 h)	Reduction (%)	µ (24-48 h)	Reduction (%)	µ (48-72 h)	Reduction (%)
Control	0.08930		0.06392		0.05923
0.10	0.09089	-1.8	0.06287	1.6	0.05849	1.3
1.0	0.08871	0.7	0.06176	3.4	0.05966	-0.7
10	0.08943	-0.1	0.06047	5.4	0.05775	2.5
100	0.08970	-0.4	0.06260	2.1	0.05738	3.1

 

Validity criteria fulfilled:

yes

Remarks:

Control cell density increased by an aver. factor of > 16 within 2d. The mean coeff. of var. for section-by-section specific growth rates in the control cultures were < 35%. The coeff. of var. of aver. spec.growth rates during the whole test were < 7%.

Conclusions:

No significant reduction of growth rate or inhibition of yield was recorded at any of the concentrations of TDI-Urone tested.

Both the EC50 for growth rate reduction (ErC50: 0-72h) and the EC50 for yield inhibition (EyC50: 0-72h) exceeded an analytically confirmed nominal concentration of 100 mg/l.

The NOEC for growth rate reduction and yield inhibition was 100 mg/l.

Executive summary:

Pseudokirchneriella subcapitata, Fresh Water Algal Growth Inhibition Test with TDI-Urone.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 761/2009 and the ISO International Standard 8692, 2004.

The batch of TDI-Urone tested was a clear colourless powder with a purity of 97.5% active content (being a mixture of isomers) and completely soluble in test medium at the concentrations tested.

A limit test was combined with a range-finding test. Six replicates of exponentially growing algal cultures were exposed to a control and a TDI-Urone concentration of 100 mg/l in the limit test. Three replicates per test group were exposed to 0.10, 1.0 and 10 mg/l in the combined range-finding test. The total test period was 72 hours and the initial algal cell density was 10^4 cells/ml. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Analysis of the samples taken from 100 mg/l at the start, after 24 hours and at the end of the test showed that the measured concentration was stable and in agreement with nominal (101-105%).

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

No significant reduction of growth rate or inhibition of yield was recorded at any of the concentrations of TDI-Urone tested.

Both the EC50 for growth rate reduction (ErC50: 0-72h) and the EC50 for yield inhibition (EyC50: 0-72h) exceeded an analytically confirmed nominal concentration of 100 mg/l.

The NOEC for growth rate reduction and yield inhibition was 100 mg/l.

Description of key information

Both the EC50 for growth rate reduction (ErC50: 0-72h) and the EC50 for yield inhibition (EC50: 0-72h) exceeded an

analytically confirmed nominal concentration of 100 mg/l.
The NOEC for growth rate reduction and yield inhibition was 100 mg/l.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 761/2009 and the ISO International Standard 8692, 2004.

The batch of TDI-Urone tested was a clear colourless powder with a purity of 97.5% active content (being a mixture of isomers) and completely soluble in test medium at the concentrations tested.

A limit test was combined with a range-finding test. Six replicates of exponentially growing algal cultures were exposed to a control and a TDI-Urone concentration of 100 mg/l in the limit test. Three replicates per test group were exposed to 0.10, 1.0 and 10 mg/l in the combined range-finding test. The total test period was 72 hours and the initial algal cell density was 10^4 cells/ml. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Analysis of the samples taken from 100 mg/l at the start, after 24 hours and at the end of the test showed that the measured concentration was stable and in agreement with nominal (101-105%).

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

No significant reduction of growth rate or inhibition of yield was recorded at any of the concentrations of TDI-Urone tested.

Both the EC50 for growth rate reduction (ErC50: 0-72h) and the EC50 for yield inhibition (EyC50: 0-72h) exceeded an analytically confirmed nominal concentration of 100 mg/l.

The NOEC for growth rate reduction and yield inhibition was 100 mg/l.