3-nitro-p-toluic 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

Study period:

2009-08-17 to 2009-08-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed OECD study with GLP

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

Test material

Test material information

Constituent 1

Reference substance name:

96-08-0

IUPAC Name:

96-08-0

Details on test material:

- Analytical purity: 98.23 % (w/w)
- Expiration date of the lot/batch: 2011-01-19

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 6.40 - 16.0 - 40.0 - 100 - 250 mg/L
- Sampling method: The concentration of the test item was analytically determined from all test concentrations and the control after 0 h (test start) and 72 h (test end). Samples at start and end of the test 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 250 mg/L was freshly prepared with dilution water and treated with ultrasound for 15 min at room temperature.
- Eluate: Dilution water
- Differential loading: 6.40 - 16.0 - 40.0 - 100 - 250 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 °C, Min: 21 °C, Max: 23 °C

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
250 7.14 6.97
100 7.76 7.57
40.0 7.83 8.64
16.0 7.98 8.82
6.40 8.04 9.02
Control 8.14 9.22

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Concentrations and Recovery Rates of Nitro-p-tolylsäure

Sampling date 2009-08-17
0 h 2009-08-20
72 h
Start of analysis 2009-08-17 2009-08-20
Nominal concentration Nitro-p-tolylsäure
Test item
Measured
conc. RR
Measured
conc. RR

[mg/L] [mg/L] [%] [mg/L] [%]
250 224 90 227 91
100 89.9 90 90.2 90
40.0 36.0 90 36.6 92
16.0 14.4 90 14.5 91
6.40 5.77 90 5.93 93
Control < LOQ — < LOQ —

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 1492333 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] Rate-related inhibition

[%] Yield Inhibition


[%]
100 7 29
10 -3 -16
1 -11 -70

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

84.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: (74.8 - 95.7)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

150 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (144 - 156)

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: 0.695 (CI 0.657 - 0.729) mg/L
Yield Inhibition: 0.320 (CI 0.294 - 0.358) mg/L

Reported statistics and error estimates:

The NOEC and LOEC were determined by calculation of statistical significance of growth rate and yield. One Way Analysis of Variance and DUNNETT’S test were carried out for the determination of statistically significant differences compared to control replicates. 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 Tests were 0.05. The a-value for ANOVA was a=0.05. Detection of statistically significant differences were considered negligible when inhibition values were ≤ 10 %. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions > 10 % were marked biological significant.

Any other information on results incl. tables

NOEC, LOEC, EC - values and 95 % Confidence Intervals of Nitro-p-tolylsäure(0-72 h)

                   based on nominal test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	40.0
LOEC	100
ErC10	81.1 (77.1 - 85.6)
ErC20	103 (98.8 - 108)
ErC50	150 (144 - 156)
	Inhibition of Yield
NOEC	40.0
LOEC	100
EyC10	46.3 (33.9 - 55.1)
EyC20	58.2 (50.7 - 67.3)
EyC50	84.7 (74.8 - 95.7)

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
250		1		5074		0	0	0
		2		5074		0	0	0
		3		5074		0	0	0
		Mean		5074		0	0	0
100		1		5074		41781	258052	536471
		2		5074		48444	248204	510797
		3		5074		60830	288974	522937
		Mean		5074		50352	265077	523402
40.0		1		5074		40691	254444	1224787
		2		5074		48091	288458	1448221
		3		5074		55738	311311	1507175
		Mean		5074		48173	284738	1393394
16.0		1		5074		45762	263345	1429471
		2		5074		61789	338576	1386881
		3		5074		57386	391843	1827832
		Mean		5074		54979	331255	1548061
6.40		1		5074		41404	274280	1325733
		2		5074		45869	322066	1483112
		3		5074		63757	390273	1453461
		Mean		5074		50343	328873	1420769
Control		1		5074		45332	281143	1390772
		2		5074		45115	340130	1465355
		3		5074		41753	277109	1384248
		4		5074		53930	346514	1763425
		5		5074		45348	314534	1554677
		6		5074		54184	285498	1395519
		Mean		5074		47610	307488	1492333

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]		[%]
250	1		n.a.	100.00		-5074	100.00
	2		n.a.	100.00		-5074	100.00
	3		n.a.	100.00		-5074	100.00
	Mean	(+)	n.a.	100.00	(+)	-5074	100.00
100	1		1.554	17.94		531397	64.27
	2		1.537	18.81		505723	66.00
	3		1.545	18.39		517863	65.18
	Mean	(+)	1.545	18.38	(+)	518328	65.15
40.0	1		1.829	3.41		1219713	17.99
	2		1.885	0.46		1443147	2.97
	3		1.898	-0.24		1502101	-1.00
	Mean	(-)	1.870	1.21	(-)	1388320	6.65
16.0	1		1.880	0.69		1424397	4.23
	2		1.870	1.22		1381807	7.09
	3		1.962	-3.64		1822758	-22.56
	Mean	(-)	1.904	-0.58	(-)	1542987	-3.75
6.40	1		1.855	2.02		1320659	11.20
	2		1.893	0.04		1478038	0.62
	3		1.886	0.40		1448387	2.61
	Mean	(-)	1.878	0.82	(-)	1415695	4.81
Control	1		1.871			1385698
	2		1.889			1460281
	3		1.870			1379174
	4		1.950			1758351
	5		1.908			1549603
	6		1.872			1390445
	Mean		1.893			1487259

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	2.190	1.825	1.599	1.871	0.298	15.9	17.9
	2	2.185	2.020	1.461	1.889	0.380	20.1
	3	2.108	1.893	1.609	1.870	0.250	13.4
	4	2.364	1.860	1.627	1.950	0.376	19.3
	5	2.190	1.937	1.598	1.908	0.297	15.6
	6	2.368	1.662	1.587	1.872	0.431	23.0
			Mean	1.893
			± SD	0.03
			CV [%]	1.67

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

n this study Nitro-p-tolylsäure was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h with the following effect values: The EC50-values with 95 % confidence intervals for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 150 (144 – 156) and 84.7 (74.8 – 95.7) mg/L, respectively. All effect levels are given based on nominal concentrations of Nitro-p-tolylsäure

Executive summary:

The toxicity of Nitro-p-tolylsäure to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201. The aim of the study was the determination of the NOEC, LOEC, EC₁₀-, EC₂₀- and EC₅₀-values of 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 10³cells/mL. Five concentration levels were tested in a geometrical series with a dilution factor of 2.5, nominal: 6.40 - 16.0 - 40.0 - 100 - 250 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 Nitro-p-tolylsäurewere analysed at all concentration levels after 0 and 72 h via HPLC analysis. The measured concentrations of Nitro-p-tolylsäureat test start were 90 % of the nominal values for all test concentrations. At the end of the test Nitro-p-tolylsäuregave recoveries of 90 – 93 % of the nominal values. All effect values are given based on nominal concentrations of the test item.

Table 1:    NOEC, LOEC, EC - values and 95 % Confidence Intervals of Nitro-p-tolylsäure(0-72 h)

                   based on nominal test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	40.0
LOEC	100
ErC10	81.1 (77.1 - 85.6)
ErC20	103 (98.8 - 108)
ErC50	150 (144 - 156)
	Inhibition of Yield
NOEC	40.0
LOEC	100
EyC10	46.3 (33.9 - 55.1)
EyC20	58.2 (50.7 - 67.3)
EyC50	84.7 (74.8 - 95.7)