2-methyl-p-phenylenediamine sulfate

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:

From Oct. 15, 2012 to Oct. 25, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed guideline, GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to OECD and German principles of GLP

Test material

Test material information

Constituent 1

Reference substance name:

2-methyl-p-phenylenediamine sulfate

EC Number:

210-431-8

EC Name:

2-methyl-p-phenylenediamine sulfate

Cas Number:

615-50-9

Molecular formula:

C7H10N2.H2O4S

IUPAC Name:

2-methylbenzene-1,4-diamine; sulfuric acid

Constituent 2

Reference substance name:

toluene-2,5-diamine sulfate

IUPAC Name:

toluene-2,5-diamine sulfate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: Toluene-2,5-diamine sulfate (Code: 95353654)
- TSIN: Not reported
- Substance type: Pure active substance
- Physical state: White to light pink or light grey powder
- Stability under test conditions: Not reported
- Storage condition of test material: Room temperature, protected from moisture and light
- Water solubility: 5.03 g/L

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 1.0, 2.20, 4.84, 10.6 and 23.4 mg/L
- Sampling period: The concentrations of test material were analysed at the beginning (0 hours) and after 6, 12, 18, 24, 48 and 72 hours in all concentration levels of the test material and the control.
- Sampling method: Sampling was performed in the following manner:
(i) At 0 hour: For the fresh media, samples of the freshly prepared test solutions (without algae) were taken and analysed.
(ii) After 6, 12 and 18 hours: Samples were taken from additionally prepared replicates (with algae) per concentration level and control.
(iii) After 24, 48 and 72 hours: Samples were taken directly from the test replicates.
1 mL per replicate was pooled for each treatment and from this pool 2 x 0.8 mL samples were taken for analysis.
- Sample storage: Retention samples for possible re-analyses were stored at room temperature.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 32 mg/L of stock solution was freshly prepared with dilution water. The dilution water was prepared in accordance with OECD guideline 201.
- Dispersion method: The stock solution was treated with ultrasound for 2 min at room temperature.
- Application of test solution: Test solution application was carried out by adding appropriate volumes of the stock solution to the test replicates.
- Controls: Prepared using dilution water without test material

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- Strain: CHODAT SAG 86.81
- Source: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universitat Gottingen, Nikolausberger Weg 18, 0-37073 Gottingen
- Age of inoculum: 3 -4 days old pre culture
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted 35-70 µE. m (-2). s(-1) for 24 h/d. Nutrient medium Z according to LOTTGE et al. (1994) was used as culture medium.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

4 - 7 days (recovery phase in fresh untreated test medium after 72 h of exposure period)

Test conditions

Hardness:

0.24 mmol Ca+Mg/L (nominal hardness)

Test temperature:

21 - 24°C, controlled at ± 2 °C (actual: 22-23°C, mean value: 22.5°C; measured by hygrothermograph)

Temperature measured continuously was determined to be within the acceptable limits.

pH:

7.91-8.21 (pH range from 0 to 72 hours; measured by pH-Meter)

pH values were determined to be within the acceptable limits.

Nominal and measured concentrations:

Nominal concentration: 0, 1, 2.2, 4.84, 10.6 and 23.4 mg/L
Time weighed average measured concentrations (0 – 72 hours): 0, 0.14, 0.31, 0.62, 1.25 and 2.47 mg/L. The percent of nominal of test material were in the range of 98 to 109 % at test start and decreased as expected to the range of < LOQM to 1 % at the test end of the exposure.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: Closed (sealed with cotton wool plugs)
- Material, fill volume: Sterile 250 mL Erlenmeyer flasks containing 100 mL of test solution
- Aeration: No
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Initial cells density: 4332 cells/mL (mean of replicates)
- Control end cells density: 877721 cells/mL (mean of replicates)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes, OECD media.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was prepared in accordance with standard OECD guideline 201. Detailed composition is provided in the study report.
- Ca/Mg ratio: CaCl2.2H2O: 18 mg/L/ (∑MgCl2.6H2O: 12 mg/L,MgSO4.7H2O: 15 mg/L)
- Hardness: 0.24 mmol Ca+Mg/L
No information on any other water parameters was provided in the study report.
- Culture medium different from test medium: Yes, Nutrient medium Z according to LUTTGE et al. (1994) was used as the culture medium.
- Intervals of water quality measurement: The pH-value at the beginning of the test was measured from one additional replicate of each concentration and control. At the end it was measured from pooled replicates per concentration and control. The room temperature was measured continuously by a hygrothermograph.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: 24 hours/day light
- Light intensity and quality: 60 - 120 µE/m(2)/sec

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell density: The cell density was measured at the beginning of the test (after application) and every 24 hours via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water with test material at the appropriate concentration but without algae was used as background signal
- Microscopic evaluation: Microscopic evaluation of cells was carried out at the start and end of the incubation. The cells were checked for unusual cell shapes, color differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for nominal test concentrations: 2.2
- Range finding study: Yes. A non-GLP range finding study was performed out at 0.1, 1, 10 and 100 mg/L (nominal) concentrations (with batch R0019281).
- Results used to determine the conditions for the definitive study: Growth rate inhibition: 100%; Yield inhibition: 100% at 100 mg/L (highest concentration tested). Based on the results of range finding study 5 concentrations were selected for main test.
Refer to ‘Table 2’ under ‘Any other information on results incl. tables’ section below.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.653 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 0.61 – 0.7 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.31 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

act. ingr.

Basis for effect:

other: inhibition of yield

Remarks on result:

other: 0.29 – 0.33 mg/L

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 7.5 - 8.4

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 4.0 - 5.1

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: inhibition of yield

Remarks on result:

other: 3.7 - 4.2

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: inhibition of yield

Remarks on result:

other: 2.0 - 2.5

Details on results:

For details on cell density, see ‘Table 3’ below in the ‘Any other information on results incl. tables’ section.
- Exponential growth in the control: Yes, after 72 hours of exposure, mean cell density in control group was 877721 cells/mL. This was an increase of 203 times over the initial inoculation density.
- Microscopic evaluation: Microscopic evaluation of the cells at start and end of the incubation period revealed no morphological abnormalities.
- Any stimulation of growth found in any treatment: No, for details on growth rate, see 'Table 4' below in the ‘Any other information on results incl. tables’ section.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid: Yes, the results of positive control were in the valid range.
- EyC50 (Yield): 0.324 mg/L; 95% CI: 0.304-0.347 (Valid range: 0.319 ± 0.140)
- ErC50 (Growth rate): 0.54 mg/L; 95% CI: 0.528-0.551 (Valid range: 0.686 ± 0.470)

Reported statistics and error estimates:

EC10, EC20 and EC50 values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated via non linear regression according to BRUCE & VERSTEEG (1992).

Any other information on results incl. tables

Recovery of algal cells

After 72hours algae were transferred from the three highest concentrations (time weighed average measured concentrations: 0.62 – 2.47 mg/L) and the control to fresh untreated medium and allowed to grow for further 4 – 7 days under test conditions.

The test material effect was observed to be reversible at these concentrations. Therefore, there is potential for recovery following exposure up to 2.47 mg/L (highest test concentration).

Table 2: Results of range finding test (0 - 72 hours)

Nominal Test material Concentration [mg/L]	Growth Rate Inhibition [%]	Yield Inhibition [%]
100	100	100
10.0	51	94
1.00	- 9	- 60
0.1	- 7	- 41

2 replicates per concentration level, 4 replicates for the control

Table 3: Effect of Toluene-2,5-Diamine Sulfate on Cell densities of green algae (Desmodesmus subspicatus) during a 72 hours exposure (Study # 66353)

Time weighed average measured concentrations [mg/L]	Replicate No.	Cell density [cells/mL]
		0 hour	24 hours	48 hours	72 hours
2.47	1	4332	< LOQ	< LOQ	2753
	2	4332	3621	< LOQ	2800
	3	4332	< LOQ	< LOQ	2734
	Mean	4332	n.a.	n.a.	2762
1.25	1	4332	2916	8187	15930
	2	4332	4489	10357	20380
	3	4332	< LOQ	10417	21837
	Mean	4332	n.a.	9654	19382
0.62	1	4332	11851	79164	301075
	2	4332	11405	56995	273814
	3	4332	11512	68049	297464
	Mean	4332	11589	68069	290784
0.31	1	4332	14848	147938	764500
	2	4332	17352	151411	899137
	3	4332	16660	156298	893885
	Mean	4332	16287	151882	852507
0.14	1	4332	24378	182483	994808
	2	4332	21399	169519	970875
	3	4332	16880	170400	920932
	Mean	4332	20886	174134	962205
Control	1	4332	22937	160474	850239
	2	4332	28498	171249	952766
	3	4332	20494	164343	884016
	4	4332	26287	179433	927889
	5	4332	25082	171859	801869
	6	4332	22299	159110	849547
	Mean	4332	24266	167745	877721

n.a. = Not Applicable

LOQ = Limit of quantification (2280 cells/mL)

Table 4: Inhibition of yield and growth rate evaluation after 72 hours of exposure (Study # 66353)

Time weighed average measured concentrations	Replicate	Growth rate		Growth rate inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
2.47	1		-0.151	100		-1579	100
	2		-0.145	100		-1532	100
	3		-0.153	100		-1598	100
	Mean	(+)	-0.150	100	(+)	-1570	100
1.25	1		0.434	75.5		11598	98.7
	2		0.516	70.8		16048	98.2
	3		0.539	69.5		17505	98.0
	Mean	(+)	0.496	72.0	(+)	15050	98.3
0.62	1		1.41	20.1		296743	66.0
	2		1.38	21.9		269482	69.2
	3		1.41	20.4		293132	66.4
	Mean	(+)	1.40	20.8	(+)	286452	67.2
0.31	1		1.72	2.57		760168	13.0
	2		1.78	-0.49		894805	-2.45
	3		1.78	-0.38		889553	-1.85
	Mean	(-)	1.76	0.57	(-)	848175	2.89
0.14	1		1.81	-2.39		990476	-13.4
	2		1.80	-1.93		966543	-10.7
	3		1.79	-0.94		916600	-4.95
	Mean	(-)	1.80	-1.75	(-)	957873	-9.68
Control	1		1.76			845907
	2		1.80			948434
	3		1.77			879684
	4		1.79			923557
	5		1.74			797537
	6		1.76			845215
	Mean		1.77			873389

Negative inhibitions = Increase of growth

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 72 h EC50 value of Toluene-2,5-Diamine sulphate to Desmodesmus subspicatus for inhibition of growth rate and inhibition of yield were 0.653 and 0.31 mg/L respectively, based on geometric mean measured concentrations, and 8.0 and 4.0 mg/l based on nominal concentrations.

Executive summary:

The toxicity of Toluene-2,5-Diamine sulphate to Desmodesmus subspicatus was determined in a 72 hours algae growth inhibition test, following OECD 201 guideline under static conditions. 

The nominal test concentrations were 0 (control), 1.0, 2.20, 4.84, 10.6 and 23.4 mg/L. Time weighed average measured concentrations were 0, 0.14, 0.31, 0.62, 1.25 and 2.47 mg/L.The percent of nominal of test material were in the range of 98 to 109 % at test start and decreased as expected to the range of < LOQ_M to 1 % at the test end of the exposure.

Endpoints evaluated were inhibition of growth rate and inhibition of yield over a period of 72 hours. 3 replicates was tested for each test concentration and 6 replicates for control.

The test material was found to inhibit the growth of freshwater green alga after 72 hours of exposure with the following effects :

1. Based on geometric mean measured concentrations):

EyC50: 0.31 mg/L (based on inhibition of yield),

ErC50: 0.653 mg/L (based on inhibition of growth rate)

2. Based on nominal concentrations:

EyC50: 4.0 mg/L (based on inhibition of yield),

ErC50: 8.0 mg/L (based on inhibition of growth rate)

After 72 hours algae were transferred from the three highest concentrations (time weighed average measured: 0.62 – 2.47 mg/L) and the control to fresh untreated medium and allowed to grow for further 4 – 7 days under test conditions. The test material effect was observed to be reversible at all test concentrations following 72 h exposure. Therefore, there is potential for recovery following exposure up to 2.47 mg/L (highest test concentration).

This toxicity test is classified as acceptable and satisfies the guideline requirements for the OECD Guideline 201.