2,4-xylidine

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:

2010

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,4-xylidine

EC Number:

202-440-0

EC Name:

2,4-xylidine

Cas Number:

95-68-1

Molecular formula:

C8H11N

IUPAC Name:

2,4-dimethylaniline

Details on test material:

- Name of test material (as cited in study report): m-Xylidin
- Molecular formula: C8H11N
- Molecular weight: 121.18 g/mol
- Physical state: Liquid, light-brown

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentrations of the test item were analytically determined from all test concentrations and the control after 0 h (test start) and 72 h (test end).
- Sampling method: Separate samples were prepared at test start. At end of the test samples were taken directly from the test replicates.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until start of analysis, if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 100 mg/L was freshly prepared with dilution water and agitated.
- Eluate: Dilution water
- Differential loading: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L
- Controls: Six replicates without test item were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.


ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Not measured

Test temperature:

Mean: 22.5 °C, Min: 22 °C, Max: 23 °C

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
100 7.86 7.54
50.0 7.84 7.81
25.0 7.82 7.95
12.5 7.83 8.07
6.25 7.82 8.31
Control 7.90 8.22

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to Any other information on materials and methods inc. tables

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: 5074 cells/mL
- Control end cells density: Mean 1330129 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Test (0-72 h)

Nominal
Test Item Concentration
[mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
100 100 100
10 1 5
1 0 1
0.1 2 8
0.01 1 6

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

28.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (27.5 - 29.9)

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
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: Yes

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.614 (CI 0.551 - 0.679) mg/L
Yield Inhibition: 0.333 (CI 0.318 - 0.350) mg/L

Reported statistics and error estimates:

NOEL/LOEL was determined by calculation of statistical significance of growth rates and yield. One Way Analysis of Variance (ANOVA) and DUNNETT’s test was used for NOEL/LOEL calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) was =0.05. Equal Variance test failed for growth rates. Transformation of the growth rates (y = y squared) was carried out.
Detection of statistically significant differences were considered negligible when inhibition values were ≤ 10 %. These findings were marked “biologically not significant”.
EL10-, EL20- and EL50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals EL10-, EL20- and EL50-values were carried out using standard procedures.

Any other information on results incl. tables

NOEC, LOEC and EC - values of m-Xylidin(0-72 h)

                   based on nominal test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	12.5
LOEC	25.0
ErC10	16.1 (14.7 – 17.6)
ErC20	20.0 (18.7 – 21.1)
ErC50	28.6 (27.5 – 29.9)
	Inhibition of Yield
NOEC	6.25
LOEC	12.5
EyC10	9.35 (8.28 – 10.6)
EyC20	11.3 (10.2 – 12.3)
EyC50	15.5 (14.4 – 16.8)

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
100		1		5074		12060	6862	2274
		2		5074		12409	6989	2036
		3		5074		12798	8055	1929
		Mean		5074		12422	7302	2080
50.0		1		5074		18907	12663	7936
		2		5074		18223	14176	10129
		3		5074		23631	13106	7017
		Mean		5074		20254	13315	8361
25.0		1		5074		35619	85733	224814
		2		5074		29871	81568	164814
		3		5074		31622	78821	143982
		Mean		5074		32371	82041	177870
12.5		1		5074		43540	257035	980206
		2		5074		38702	238676	871650
		3		5074		45734	253649	939792
		Mean		5074		42659	249787	930549
6.25		1		5074		44348	299585	1445601
		2		5074		45808	242439	1458762
		3		5074		48600	302094	1440787
		Mean		5074		46252	281373	1448383
Control		1		5074		42544	304337	1320805
		2		5074		40248	230664	1141504
		3		5074		48149	278515	1428393
		4		5074		43516	249840	1351149
		5		5074		46841	258278	1397057
		6		5074		43421	290442	1341863
		Mean		5074		44120	268679	1330129

Evaluation after 72 h

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Nominal test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		-0.268	100		-2800	100
	2		-0.304	100		-3038	100
	3		-0.322	100		-3145	100
	Mean	(+)	-0.298	100	(+)	-2994	100
50.0	1		0.149	92.0		2862	99.8
	2		0.230	87.6		5055	99.6
	3		0.108	94.2		1943	99.9
	Mean	(+)	0.16	91.2	(+)	3287	99.8
25.0	1		1.26	31.9		219740	83.4
	2		1.16	37.5		159740	87.9
	3		1.12	39.9		138908	89.5
	Mean	(+)	1.18	36.4	(+)	172796	87.0
12.5	1		1.76	5.44		975132	26.4
	2		1.72	7.55		866576	34.6
	3		1.74	6.20		934718	29.5
	Mean	(+)*	1.74	6.40	(+)	925475	30.2
6.25	1		1.88	-1.54		1440527	-8.71
	2		1.89	-1.70		1453688	-9.71
	3		1.88	-1.48		1435713	-8.35
	Mean	(-)	1.89	-1.57	(-)	1443309	-8.92
Control	1		1.85			1315731
	2		1.81			1136430
	3		1.88			1423319
	4		1.86			1346075
	5		1.87			1391983
	6		1.86			1336789
	Mean		1.86			1325055

Section-by-Section and Average Specific Growth Rates of the Control Group
                   (0 – 72 h)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 h	24 - 48 h	48 - 72 h
Control	1	2.13	1.97	1.47	1.85	0.344	18.5	15.9
	2	2.07	1.75	1.60	1.81	0.241	13.4
	3	2.25	1.76	1.64	1.88	0.326	17.3
	4	2.15	1.75	1.69	1.86	0.251	13.5
	5	2.22	1.71	1.69	1.87	0.303	16.2
	6	2.15	1.90	1.53	1.86	0.310	16.7
				Mean	1.86
				SD ±	0.03
				CV [%]	1.42

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study m-Xylidin was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h. The NOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 12.5 and 6.25 mg/L, respectively. The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 25.0 and 12.5 mg/L, respectively. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 28.6 (27.5 – 29.9) and 15.5 (14.4 – 16.8) mg/L, respectively.
All effect levels are given based on nominal concentrations of m-Xylidin.

Executive summary:

The toxicity of m-Xylidin to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 over a period of 72 h.

The study was conducted under static conditions with an initial cell density of approximately   2 – 5 x 10³cells/mL. Based on a preliminary test 5 concentration levels were tested in a geometrical series with a dilution factor of 2, nominal: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of m-Xylidinwere analysed at all concentration levels after 0 and 72 h via HPLC analysis. The measured concentrations of m-Xylidinat test start were in the range of 100 – 103 % of the nominal values. At the end of the test m-Xylidingave recoveries of 95 – 98 % of the nominal values. All effect values are given based on nominal concentrations of the test item.

Table 1:    NOEC, LOEC and EC - values of m-Xylidin(0-72 h)

                   based on nominal test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	12.5
LOEC	25.0
ErC10	16.1 (14.7 – 17.6)
ErC20	20.0 (18.7 – 21.1)
ErC50	28.6 (27.5 – 29.9)
	Inhibition of Yield
NOEC	6.25
LOEC	12.5
EyC10	9.35 (8.28 – 10.6)
EyC20	11.3 (10.2 – 12.3)
EyC50	15.5 (14.4 – 16.8)