N,N-dimethyl-p-toluidine

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 8, 12, 18, 27, and 40.5 mg/L
- Sample storage conditions before analysis: test sample analysed soon after sampling

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test substance was directly dissolved in the OECD medium, which was filtered and final solublity was measured analytically. 250 ml of test substance was dissolved in 250 ml of OECD medium, the analytically measured concentration was 544.18 mg/l.
- Controls: OECD medium itself was used as control with no test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain:
- Source (laboratory, culture collection): Sterile, unicellular, suspension cultures of algae were obtained from Denmark Technical University, Denmark The principle culture at the DTU is obtained from Norwegian Institute for Water Research, Oslo, Norway (NIVA).
- Age of inoculum (at test initiation): 10000 cells/ml
- Method of cultivation: The growth medium used for the culturing of Pseudokirchneriella subcapitata is OECD Medium The temperatures were maintained at 21 to 24°C, controlled at ± 2°C. The Light intensity provided to the culture, it is measured each time and should be 7000 - 8000 lux at the surface and a constant light phase is maintained for the test duration. Pre-culture   Pre-culture was set up two days prior to initiation of the test. It is grown under identical exposure conditions.

ACCLIMATION
- Acclimation period: The algal inocula for a test was taken from an exponentially-growing pre-culture and mixed with the OECD medium. which was minimum for 2-3 days
- Culturing media and conditions (same as test or not): same as test conditions
- Any deformed or abnormal cells observed: None

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.8°C

pH:

Control Start 7.5 and end 8.3

Nominal and measured concentrations:

Nominal conentration: 8, 12, 18, 27, and 40.5 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml Conical glass flask
- Type (delete if not applicable): open / closed closed
- Material, size, headspace, fill volume: glass size of 100 ml head space of 40 ml
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- Initial cells density: 10000 cells/ml
- Control end cells density: 250000 cells/ml
- No. of organisms per vessel: 600000 cell
- No. of vessels per concentration (replicates): 3 replicates per concentrations
- No. of vessels per control (replicates): 3 replicates per controls
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: Not applicable


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Not adjusted
- Photoperiod: Continues light
- Light intensity and quality: 7000-8000 lux
- Salinity (for marine algae): Not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell count was done using neubaur counting chamber under microscope.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.5
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: not conduted based definitive test was conducted based on the available data

Reference substance (positive control):

yes

Remarks:

K2Cr2O7

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

23.69 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

35.65 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): None
- Unusual cell shape: None observed on control
- Colour differences: None observed on control
- Adherence to test vessels: Not adhered
- Aggregation of algal cells: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ErC50: 2.331 mg/L (Nominal concentration)

Reported statistics and error estimates:

To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (ErC) were determined.

pH and temperature:

Test Concentration(mg/L)	Experimental Flasks	pH		Temperature °C
		0 Hours	72 Hours	0 Hours	72 Hours
control	(R1)	7.5	8.3	21.8	21.8
control	(R2)	7.6	8.4	21.8	21.8
control	(R3)	7.7	8.7	21.8	21.8
8	(R1)	7.6	8.9	21.8	21.8
8	(R2)	7.7	8.1	21.8	21.8
8	(R3)	7.8	9.2	21.8	21.8
12	(R1)	7.7	8.9	21.8	21.8
12	(R2)	7.8	8.7	21.8	21.8
12	(R3)	7.8	8.8	21.8	21.8
18	(R1)	7.7	8.5	21.8	21.8
18	(R2)	7.8	8.6	21.8	21.8
18	(R3)	7.7	8.4	21.8	21.8
27	(R1)	7.6	8.5	21.8	21.8
27	(R2)	7.7	8.5	21.8	21.8
27	(R3)	7.7	8.6	21.8	21.8
40.5	(R1)	7.7	8.3	21.8	21.8
40.5	(R2)	7.7	8.0	21.8	21.8
40.5	(R3)	7.8	8.0	21.8	21.8

		DAY 0				DAY 1				DAY 2				DAY 3
Experimental Sets	TEST CONC (mg/L)	Count 1	Count 2	Average	Ln(Av)	Count 1	Count 2	Average	Ln(Av)	Count 1	Count 2	Average	Ln(Av)	Count 1	Count 2	Average	Ln(Av)	Daily Growth rate 0-1 days	Daily Growth rate 1-2 days	Daily Growth rate 2-3 days	Average Growth rate 0-3 days	Standard deviation	Mean	Coefficient of Variation	Inhibition	% Inhibition
Control 1	0.0	10000	10000	10000	9.21	40000	40000	40000	10.60	120000	80000	100000	11.51	240000	260000	250000	12.43	1.39	0.92	0.92	1.07	0.029	1.04	0.028
Control 2	0.0	10000	10000	10000	9.21	40000	30000	35000	10.46	80000	100000	90000	11.41	200000	220000	210000	12.25	1.25	0.94	0.85	1.01
Control 3	0.0	10000	10000	10000	9.21	30000	30000	30000	10.31	90000	100000	95000	11.46	230000	220000	225000	12.32	1.10	1.15	0.86	1.04
REP 1	8	10000	10000	10000	9.21	30000	30000	30000	10.31	90000	80000	85000	11.35	220000	220000	220000	12.30	1.10	1.04	0.95	1.03	0.082	0.95	0.086	0.08	8.45
REP 2	8	10000	10000	10000	9.21	30000	30000	30000	10.31	80000	100000	90000	11.41	160000	200000	180000	12.10	1.10	1.10	0.69	0.96
REP 3	8	10000	10000	10000	9.21	30000	40000	35000	10.46	100000	90000	95000	11.46	130000	140000	135000	11.81	1.25	1.00	0.35	0.87
REP 1	12	10000	10000	10000	9.21	30000	30000	30000	10.31	50000	60000	55000	10.92	130000	140000	135000	11.81	1.10	0.61	0.90	0.87	0.012	0.88	0.014	0.16	15.58
REP 2	12	10000	10000	10000	9.21	20000	20000	20000	9.90	60000	50000	55000	10.92	130000	150000	140000	11.85	0.69	1.01	0.93	0.88
REP 3	12	10000	10000	10000	9.21	20000	20000	20000	9.90	60000	60000	60000	11.00	140000	150000	145000	11.88	0.69	1.10	0.88	0.89
REP 1	18	10000	10000	10000	9.21	20000	20000	20000	9.90	50000	60000	55000	10.92	140000	130000	135000	11.81	0.69	1.01	0.90	0.87	0.007	0.86	0.008	0.17	17.13
REP 2	18	10000	10000	10000	9.21	20000	20000	20000	9.90	60000	60000	60000	11.00	120000	140000	130000	11.78	0.69	1.10	0.77	0.85
REP 3	18	10000	10000	10000	9.21	20000	20000	20000	9.90	50000	60000	55000	10.92	140000	130000	135000	11.81	0.69	1.01	0.90	0.87
REP 1	27	10000	10000	10000	9.21	20000	20000	20000	9.90	30000	20000	25000	10.13	90000	100000	95000	11.46	0.69	0.22	1.34	0.75	0.010	0.74	0.014	0.29	28.55
REP 2	27	10000	10000	10000	9.21	20000	20000	20000	9.90	30000	30000	30000	10.31	100000	90000	95000	11.46	0.69	0.41	1.15	0.75
REP 3	27	10000	10000	10000	9.21	20000	10000	15000	9.62	20000	40000	30000	10.31	90000	90000	90000	11.41	0.41	0.69	1.10	0.73
REP 1	40.5	10000	10000	10000	9.21	20000	10000	15000	9.62	30000	20000	25000	10.13	30000	40000	35000	10.46	0.41	0.51	0.34	0.42	0.043	0.36	0.118	0.65	65.15
REP 2	40.5	10000	10000	10000	9.21	20000	10000	15000	9.62	20000	20000	20000	9.90	20000	30000	25000	10.13	0.41	0.29	0.22	0.31
REP 3	40.5	10000	10000	10000	9.21	10000	10000	10000	9.21	20000	20000	20000	9.90	30000	30000	30000	10.31	0.00	0.69	0.41	0.37

Analytical concentration determination:

			0 Hours		72 Hours	Geometric mean  (mg/l)
SR. No	concentrations (mg/L)		Analytical concentrations		Analytical concentrations	Measured concentrations
1	Blank		0.00		0.00	0
2	8		7.13		4.70	5.92
3	12		10.4		6.42	8.43
4	18		15.52		10.83	13.18
5	27		22.45		15.50	18.98
6	40.5		32.45		17.35	24.9

Validity criteria fulfilled:

yes

Conclusions:

Based on the growth rate of fresh water algae the median lethal concentration EC50 was reported to be 23.69 mg/L after 72 hours of eposure period.

Executive summary:

The effect of test chemical on fresh water algae was studied by following OECD 201 guidelines using P subcapitata as test system. The mother culture was maintained in the same conditions of the test. From this preculture flask was taken and incubated for 3 days to attain the exponential growth. Cells from the exponential growth was taken for the main study. Based on the available data, main study was directly conducted with 5 test concentrations. The test substance was dissolved in the OECD medium and stirring was provided, to distribute chemical homogeniously, which was further analytically determined using UV visible spectrophotometer. Test concentrations selected for the study are 8, 12, 18, 27 and 40.5 mg/L separated with a factor of 1.5. Each test concentration was maintained in the triplicates along with control in 100 ml conical flask with 60 ml of test solution including test system. The algal cell count in each flask was 10000 cell/ ml at day 0. All the flasks were placed in an incubator and maintained for the period of 72 hours at a temperature of 21.8°C. The cell count was done using Neubauer chamber under microscope at regular 24 hours i.e., day0, day1 day 2 and day3 in all test vessels. The pH at the day 0 and day 3 was 7.5 and 8.3 respectively. The percent inhibition of cell growth at concentrations 8, 12, 18, 27, 40.5 was 8.5%, 15.58%, 17.13%, 28.55% and 65.55% respectively. All the test concentrations were analyzed for actual test substance, at 0 hours and 72 hours. which were not maintained within 80 -120% of the nominal concentrations, thus geometric mean was reported. The biomass of the control cultures have increased exponentially by a factor of 22.83 (specific growth rate 1.04 day-1) within the 72 hr period which is following the validity criteria of biomass in the control cultures of factor 16 (specific growth rate of 0.92 day-1) within the 72 hr test periods. The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hr tests) in the control cultures is9.52 %, thereby meeting the 35% validity criteria. The mean coefficient of variation of average specific growth rates during the whole test period in replicate control culture was found to be2.81 %and it must not be exceed 7% in the tests with Pseudokirchneriella subcapitata. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (ErC) were determined. thus EC50 was reported in geometric mean measured concentration of 0 hours and 72 hours. Based on outcomes of growth rates EC50 was reported to be 23.69mg/l (measured) and 35.65 mg/l (nominal). Thus chemical can be classified into aquatic chronic category 3 as per CLP classification criteria

Description of key information

Toxicity to aquatic algae:

The effect of test chemical on fresh water algae was studied by following OECD 201 guidelines using P subcapitata as test system. The mother culture was maintained in the same conditions of the test. From this preculture flask was taken and incubated for 3 days to attain the exponential growth. Cells from the exponential growth was taken for the main study. Based on the available data, main study was directly conducted with 5 test concentrations. The test substance was dissolved in the OECD medium and stirring was provided, to distribute chemical homogeniously, which was further analytically determined using UV visible spectrophotometer. Test concentrations selected for the study are 8, 12, 18, 27 and 40.5 mg/L separated with a factor of 1.5. Each test concentration was maintained in the triplicates along with control in 100 ml conical flask with 60 ml of test solution including test system. The algal cell count in each flask was 10000 cell/ ml at day 0. All the flasks were placed in an incubator and maintained for the period of 72 hours at a temperature of 21.8°C. The cell count was done using Neubauer chamber under microscope at regular 24 hours i.e., day0, day1 day 2 and day3 in all test vessels. The pH at the day 0 and day 3 was 7.5 and 8.3 respectively. The percent inhibition of cell growth at concentrations 8, 12, 18, 27, 40.5 was 8.5%, 15.58%, 17.13%, 28.55% and 65.55% respectively. All the test concentrations were analyzed for actual test substance, at 0 hours and 72 hours. which were not maintained within 80 -120% of the nominal concentrations, thus geometric mean was reported. The biomass of the control cultures have increased exponentially by a factor of 22.83 (specific growth rate 1.04 day-1) within the 72 hr period which is following the validity criteria of biomass in the control cultures of factor 16 (specific growth rate of 0.92 day-1) within the 72 hr test periods. The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hr tests) in the control cultures is9.52 %, thereby meeting the 35% validity criteria. The mean coefficient of variation of average specific growth rates during the whole test period in replicate control culture was found to be2.81 %and it must not be exceed 7% in the tests with Pseudokirchneriella subcapitata. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (ErC) were determined. thus EC50 was reported in geometric mean measured concentration of 0 hours and 72 hours. Based on outcomes of growth rates EC50 was reported to be 23.69mg/l (measured) and 35.65 mg/l (nominal). Thus chemical can be classified into aquatic chronic category 3 as per CLP classification criteria

Key value for chemical safety assessment

EC50 for freshwater algae:

23.69 mg/L

Additional information

Based on the various predicted and experimental studies from different sources, studies were reviewed for the determination of the effects of test chemical and structurally and functionally similar read across chemicals on the growth rate inhibition of algae. The studies are as mentioned below:

The effect of test chemical on fresh water algae was studied by following OECD 201 guidelines using P subcapitata as test system. The mother culture was maintained in the same conditions of the test. From this preculture flask was taken and incubated for 3 days to attain the exponential growth. Cells from the exponential growth was taken for the main study. Based on the available data, main study was directly conducted with 5 test concentrations. The test substance was dissolved in the OECD medium and stirring was provided, to distribute chemical homogeniously, which was further analytically determined using UV visible spectrophotometer. Test concentrations selected for the study are 8, 12, 18, 27 and 40.5 mg/L separated with a factor of 1.5. Each test concentration was maintained in the triplicates along with control in 100 ml conical flask with 60 ml of test solution including test system. The algal cell count in each flask was 10000 cell/ ml at day 0. All the flasks were placed in an incubator and maintained for the period of 72 hours at a temperature of 21.8°C. The cell count was done using Neubauer chamber under microscope at regular 24 hours i.e., day0, day1 day 2 and day3 in all test vessels. The pH at the day 0 and day 3 was 7.5 and 8.3 respectively. The percent inhibition of cell growth at concentrations 8, 12, 18, 27, 40.5 was 8.5%, 15.58%, 17.13%, 28.55% and 65.55% respectively. All the test concentrations were analyzed for actual test substance, at 0 hours and 72 hours. which were not maintained within 80 -120% of the nominal concentrations, thus geometric mean was reported. The biomass of the control cultures have increased exponentially by a factor of 22.83 (specific growth rate 1.04 day-1) within the 72 hr period which is following the validity criteria of biomass in the control cultures of factor 16 (specific growth rate of 0.92 day-1) within the 72 hr test periods. The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hr tests) in the control cultures is9.52 %, thereby meeting the 35% validity criteria. The mean coefficient of variation of average specific growth rates during the whole test period in replicate control culture was found to be2.81 %and it must not be exceed 7% in the tests with Pseudokirchneriella subcapitata. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (ErC) were determined. thus EC50 was reported in geometric mean measured concentration of 0 hours and 72 hours. Based on outcomes of growth rates EC50 was reported to be 23.69mg/l (measured) and 35.65 mg/l (nominal). Thus chemical can be classified into aquatic chronic category 3 as per CLP classification criteria

Based on the prediction done using the EPI Suite ECOSAR, the toxicity on green algae was predicted for test chemical. Based on effects observed in a static system, the effect concentration EC50 value for the test substance was estimated to be 14.962 mg/l for aquatic algae after exposure period of 72 hours. Based on this value, it can be concluded that the test chemical can be considered as toxic to green algae at environmentally relevant concentrations and can be considered to be classified in aquatic chronic category 3 as per the CLP classification criteria.

 

Above data further supported by the second experimental study from peer reviewed journal. Principle of this study was to determine the effect of test chemical on the population growth rate inhibition of algae. Effects on the population growth of algae was tested according to the OECD guidelines for testing chemicals. Chlorella pyrenoidosa was used a test organism. Green algae were collected from H. Oldersma from TNO (Delft, The Netherlands). The algae were cultured in Miller’s solution using a 100 ml bottle, which was renewed (1:5) at least once a week. The algal cultures were incubated on top of a series of TL tubes and were shaken by a microplate shaker at 300 rpm. 5 Nominal concentrations and 1 control were used in the study. Effects were observed in the interval of 0, 6, 24, 48 and 72 hours. Based on the growth rate inhibition of green algae Chlorella pyrenoidosa due to the test chemical exposure for 72 hours, the EC50 value was determined to be 22 mg/l. Thus, based on the EC50 value, test chemical considers to be toxic and classified in aquatic chronic category 3 as per the CLP classification criteria.

 

Similar study was conducted to determine the effect of test chemical on the population growth rate inhibition of green algae. Pseudokirchneriella subcapitata was used a test organism. Test conducted under the closed system using triplicates. Algal growth medium was used in the study. Nominal concentrations were used to determine the effects. 15000 cells/ml was used and obtained from the laboratory.Test was performed at a temperature of 24°C. After the exposure period of 48 hours, 50 % median effective concentration were determined. Based on thepopulation growth rate inhibition of green algae Pseudokirchneriella subcapitata due to the test chemical exposure for 48 hours, the EC50 value was obtained to be at 14.19 mg/l.

In the fourth weight of evidence study from experimental source, the nature of test chemical when comes in contact with the green algae was determined. Test was conducted according to the OECD guideline 201. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) was used as test organism. The stock solution (200 g/L) was prepared in acetone. Test solutions of required concentration were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. 0, 12, 25, 50, 100 and 200 mg/L, respectively concentrations were used. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of test chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Effect on the growth of algae was determined after an exposure period of 72 hrs. The median effective concentration (ErC50 value) of the test chemical on green algae was determined to be 59.2 mg/L with 95% CI of 38 mg/l to 92.4 mg/l on the basis of growth rate inhibition effects in a 72-hour study. Based on the ErC50 value, the chemical was considered likely to be hazardous to aquatic algae and can be considered to be classified in aquatic chronic 3 category as per the CLP classification criteria.

 

Thus, based on above all studies and effects observation, it was concluded that the test chemical considers to be toxic and classified in aquatic chronic category 3 as per the CLP classification criteria.