Dimethyl 2-aminoterephthalate

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:

2009-12-11 to 2009-12-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline conform study with GLP

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:

Dimethyl 2-aminoterephthalate

EC Number:

226-364-2

EC Name:

Dimethyl 2-aminoterephthalate

Cas Number:

5372-81-6

Molecular formula:

C10H11NO4

IUPAC Name:

1,4-dimethyl 2-aminobenzene-1,4-dicarboxylate

Details on test material:

- Name of test material (as cited in study report): Amino-DMT trocken
- Molecular formula: C10H11NO4
- Molecular weight: 209.20 g/mol
- Physical state: Solid, grey powder
-

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 saturated solution* of 800 mg/L was prepared with dilution water. The dispersion was shaken with 20 rpm for 48 h
at ambient temperature and filtrated with 0.45 µm RC filter.
- Eluate: Dilution water
- Differential loading: The saturated solution and further four dilution levels were tested in a geometrical series with a dilution factor 2: 6.25 - 12.5 - 25 - 50 and 100 % of the saturated solution corresponding to initial measured concentrations of 1.33 – 2.67 – 5.46 – 10.9 – 21.6 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):4 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: 21.5 °C, Min: 21 °C, Max: 22 °C

pH:

Initial measured test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
21.6 8.10 8.04
10.9 8.18 7.96
5.46 8.19 7.92
2.67 8.19 8.26
1.33 8.07 8.50
Control 8.20 8.43

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods incl. 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 690386 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)

Saturated solution
[%] Rate Related Inhibition[%] Yield Inhibition [%]
100 88 100
10 - 1 - 7
1 2 11
0.1 1 6

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.46 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (8.55 - 10.5)

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

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.600 (CI 0.545 - 0.653) mg/L
Yield Inhibition: 0.280 (CI 0.266 - 0.299) mg/L

Reported statistics and error estimates:

NOEC/LOEC 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 NOEC/LOEC 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 a=0.05.

EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals EC10-, EC20- and EC50-values were carried out using standard procedures.

Any other information on results incl. tables

NOEC, LOEC and EC - values of Amino-DMT trocken (0-72 h)

                   based on initial measured test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	1.33
LOEC	2.67
ErC10	2.82 ( 1.33 – 3.85)
ErC20	4.80 (3.94 – 5.67)
ErC50	9.46 (8.55 – 10.5)
	Inhibition of Yield
NOEC	1.33
LOEC	2.67
EyC10	1.33
EyC20	1.49 ( 1.33 – 1.90)
EyC50	2.88 (2.16 – 3.84)

Cell Densities

	Initial measured test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
21.6		1		5074		21146	12954	9236
		2		5074		21421	12926	9084
		3		5074		22712	11294	9928
		Mean		5074		21760	12391	9416
10.9		1		5074		38997	31921	31827
		2		5074		38161	30474	35779
		3		5074		36919	32298	50510
		Mean		5074		38026	31564	39372
5.46		1		5074		37644	100190	230012
		2		5074		40277	86475	186753
		3		5074		41708	127947	302525
		Mean		5074		39876	104871	239763
2.67		1		5074		36480	160308	360368
		2		5074		36816	175601	333062
		3		5074		41720	164380	340393
		Mean		5074		38339	166763	344608
1.33		1		5074		34512	209221	590488
		2		5074		39539	193170	519862
		3		5074		36316	207626	693501
		Mean		5074		36789	203339	601284
Control		1		5074		31023	161510	674350
		2		5074		26402	184183	655970
		3		5074		30125	174880	698195
		4		5074		31564	169103	708978
		5		5074		33167	174248	633629
		6		5074		30724	212022	771196
		Mean		5074		30501	179324	690386

Evaluation after 72 h

                   Statistically significant differences of growth rates and yield compared to

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

Initial measured test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
21.6	1		0.200	87.8		4162	99.4
	2		0.194	88.1		4010	99.4
	3		0.224	86.3		4854	99.3
	Mean	(+)	0.206	87.4	(+)	4342	99.4
10.9	1		0.612	62.6		26753	96.1
	2		0.651	60.2		30705	95.5
	3		0.766	53.2		45436	93.4
	Mean	(+)	0.676	58.7	(+)	34298	95.0
5.46	1		1.27	22.3		224938	67.2
	2		1.20	26.6		181679	73.5
	3		1.36	16.8		297451	56.6
	Mean	(+)	1.28	21.9	(+)	234689	65.8
2.67	1		1.42	13.2		355294	48.2
	2		1.40	14.8		327988	52.1
	3		1.40	14.4		335319	51.1
	Mean	(+)	1.41	14.1	(+)	339534	50.5
1.33	1		1.59	3.14		585414	14.6
	2		1.54	5.74		514788	24.9
	3		1.64	-0.130		688427	-0.450
	Mean	(-)	1.59	2.92	(-)	596210	13.0
Control	1		1.63			669276
	2		1.62			650896
	3		1.64			693121
	4		1.65			703904
	5		1.61			628555
	6		1.68			766122
	Mean		1.64			685312

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	1.81	1.65	1.43	1.63	0.192	11.7	16.1
	2	1.65	1.94	1.27	1.62	0.337	20.8
	3	1.78	1.76	1.38	1.64	0.223	13.6
	4	1.83	1.68	1.43	1.65	0.199	12.1
	5	1.88	1.66	1.29	1.61	0.296	18.4
	6	1.80	1.93	1.29	1.68	0.338	20.2
				Mean	1.64
				SD ±	0.02
				CV [%]	1.40

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study Amino-DMT trocken 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 1.33 mg/L. The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 h were 2.67 mg/L. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 9.46 (8.55 – 10.5) and 2.88 (2.16 – 3.84) mg/L, respectively.
All effect levels are given based on initial measured concentrations of Amino-DMT trocken.

Executive summary:

The toxicity of Amino-DMT trocken to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201.

The study was conducted under static conditions with an initial cell density of approximately  2 – 5 x 10³cells/mL. Based on a preliminary test, a definitive test with the saturated solution and further four dilution levels were tested in a geometrical series with a dilution factor of 2, nominal: 6.25 - 12.5 - 25 - 50 - 100 % of the saturated solution corresponding to initial measured test item concentrations of 1.33 – 2.67 – 5.46 – 10.9 – 21.6 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 Amino-DMT trocken were analysed at all concentration levels after 0 and 72 h via HPLC analysis. The measured concentrations of Amino-DMT trocken at test start were in the range of 99 – 101 % of the saturated solution. At the end of the test Amino-DMT trocken gave recoveries of 82 – 91 % of the initial measured concentrations. All effect values are given based on initial measured concentrations of the test item.

NOEC, LOEC and EC - values of Amino-DMT trocken (0-72 h)

                   based on initial measured test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	1.33
LOEC	2.67
ErC10	2.82 ( 1.33 – 3.85)
ErC20	4.80 (3.94 – 5.67)
ErC50	9.46 (8.55 – 10.5)
	Inhibition of Yield
NOEC	1.33
LOEC	2.67
EyC10	1.33
EyC20	1.49 ( 1.33 – 1.90)
EyC50	2.88 (2.16 – 3.84)