2,5-bis(p-toluidino)terephthalic acid

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to relevant guidelines and compliant to GLP. Results are well documented and plausible.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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:

GLP according to German Chemikaliengesetz and directive 88/320/EEC

Test material

Test material information

Constituent 1

Reference substance name:

2,5-bis(p-toluidino)terephthalic acid

EC Number:

233-651-6

EC Name:

2,5-bis(p-toluidino)terephthalic acid

Cas Number:

10291-28-8

Molecular formula:

C22H20N2O4

IUPAC Name:

2,5-bis[(4-methylphenyl)amino]benzene-1,4-dicarboxylic acid

Details on test material:

- Name of test material (as cited in study report): Toluidino acid
- Molecular formula: C22H20N2O4
- Molecular weight: 376.42 g/mol
- Physical state: Solid, violett powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: A saturated solution was prepared.
- Sampling method: The limit concentration and the control were analytically verified at the beginning and the end of the test via HPLC-DAD (diode array detection) analysis. Samples at test start and end 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 was prepared. A dispersion of 20 mg/L prepared in dilution water was shaken with 20 rpm for 48 h at ambient temperature and filtrated with 0.45 µm RC filter.
- Eluate: Dilution water
- Differential loading: Limit test
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None, filtration

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:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

Not measured

Test temperature:

Mean: 23 °C, Min: 22 °C, Max: 24 °C

pH:

Geometric mean measured test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
4.10 8.28 8.38
Control 8.07 8.56

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

See "Any other information on materials and methods"!

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 1184583 cells/mL
- No. of vessels per concentration (replicates): 6
- 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)

See "Any other information on materials and methods"

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.1 other: mg/L (represents the saturation concentration)

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and biomass

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:

All calculations were based on the fluorescence values of each replicate.

Any other information on results incl. tables

NOEC, LOEC, EC - values and 95 % Confidence Intervals of Toluidino acid
(0-72 h)

                   based on geometric mean measured test item concentration [mg/L]

                               

	Rate-related Inhibition
NOEC	4.10
LOEC	> 4.10
ErC10	> 4.10
ErC20	> 4.10
ErC50	> 4.10
	Inhibition of Yield
NOEC	4.10
LOEC	> 4.10
EyC10	> 4.10
EyC20	> 4.10
EyC50	> 4.10

Cell Densities

	Geometric mean measured test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
4.10		1		5074		40531	302656	1400997
		2		5074		45398	279520	1349681
		3		5074		48493	304796	1471779
		4		5074		44787	326256	1507884
		5		5074		51986	299466	1476191
		6		5074		47300	317736	1344097
		Mean		5074		46416	305072	1425105
Control		1		5074		43479	294526	1381862
		2		5074		38571	302849	1134259
		3		5074		35976	221624	1125042
		4		5074		43471	240570	1072517
		5		5074		39473	217700	1147195
		6		5074		43639	295731	1246620
		Mean		5074		40768	262167	1184583

Evaluation after 72 h

                              

Geometric mean measured test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
4.10	1		1.87	-3.14		1395923	-18.4
	2		1.86	-2.46		1344607	-14.0
	3		1.89	-4.05		1466705	-24.4
	4		1.90	-4.49		1502810	-27.4
	5		1.89	-4.10		1471117	-24.7
	6		1.86	-2.38		1339023	-13.5
	Mean		1.88	-3.44		1420031	-20.4
Control	1		1.87			1376788
	2		1.80			1129185
	3		1.80			1119968
	4		1.79			1067443
	5		1.81			1142121
	6		1.84			1241546
	Mean		1.82			1179509

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	2.15	1.91	1.55	1.87	0.304	16.2	16.5
	2	2.03	2.06	1.32	1.80	0.418	23.2
	3	1.96	1.82	1.63	1.80	0.168	9.32
	4	2.15	1.71	1.50	1.79	0.333	18.6
	5	2.05	1.71	1.66	1.81	0.213	11.8
	6	2.15	1.91	1.44	1.84	0.363	19.8
				Mean	1.82
				SD ±	0.03
				CV [%]	1.68

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study Toluidino acid was found not to inhibit the growth rate of the freshwater green alga Desmodesmus subspicatus in a limit test after 72 h at the saturation concentration of 4.10 mg/L. A slight increase of growth was observed.

Executive summary:

The toxicity of Toluidino acid to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr .U. Noack-Laboratorien in 31157 Sarstedt, Germany from September 28 to October 01, 2009. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h.

The study was conducted under static conditions with an initial cell density of approximately  2 – 5 x 10³ cells/mL. Based on a preliminary range finding test, a limit test with the saturated solution was carried out. Six replicates were tested for limit concentration and control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of Toluidino acid were analysed at the limit concentration at test start and test end via HPLC-DAD analysis. The measured concentrations of the test item at test start and end were 5.12 and 3.29 mg/L, respectively. All effect values are given based on geometric mean measured concentration of the test item. Toluidino acid caused a slight stimulation of growth.

NOEC, LOEC, EC - values and 95 % Confidence Intervals of Toluidino acid
(0-72 h)

                   based on geometric mean measured test item concentration [mg/L]

                               

	Rate-related Inhibition
NOEC	4.10
LOEC	> 4.10
ErC10	> 4.10
ErC20	> 4.10
ErC50	> 4.10
	Inhibition of Yield
NOEC	4.10
LOEC	> 4.10
EyC10	> 4.10
EyC20	> 4.10
EyC50	> 4.10