1-chloro-2,5-dimethoxy-4-nitrobenzene

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:

2009-10-16 to 2009-10-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented GLP and guideline study.

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 (incl. QA statement)

Remarks:

GLP compliance statement and certificate included in full study report.

Test material

Test material information

Constituent 1

Reference substance name:

1-chloro-2,5-dimethoxy-4-nitrobenzene

EC Number:

230-086-7

EC Name:

1-chloro-2,5-dimethoxy-4-nitrobenzene

Cas Number:

6940-53-0

Molecular formula:

C8H8ClNO4

IUPAC Name:

1-chloro-2,5-dimethoxy-4-nitrobenzene

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: 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 saturated solution* was prepared. A dispersion of 1440 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: The saturated solution and further four dilution levels were tested in a geometrical series with a dilution factor √10: 1.0 - 3.2 - 10 - 32 and 100 % of the saturated solution corresponding to initial measured concentrations of 0.106 – 0.323 – 0.974 – 3.26 – 9.92 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: 23.5 °C, Min: 23 °C, Max: 25 °C

pH:

Initial measured test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
9.92 8.12 8.13
3.26 8.12 8.35
0.974 8.10 8.32
0.323 8.09 8.33
0.106 8.06 8.34
Control 7.96 8.34

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 492829 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

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.26 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

9.92 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.46 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (3.26 - 9.92)

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.26 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

9.92 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: CI: 3.26 - 9.92

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: No
- Colour differences: Not reported
- Flocculation: No data
- Adherence to test vessels: No
- Aggregation of algal cells: yes
- Other: Not reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not reported

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:

EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 h were calculated by linear dose-response regression. Calculation of the confidence intervals were estimated empirically.

NOEC, LOEC 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.

Any other information on results incl. tables

NOEC, LOEC and EC - values of 1-Chlor-2,5-dimethoxy-4-nitrobenzol trocken (0-72 h)

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

                               

	Rate-related Inhibition
NOEC	3.26
LOEC	9.92
ErC10	4.12 (3.26 – 9.92)
ErC20	4.60 (3.26 – 9.92)
ErC50	6.46 (3.26 – 9.92)
	Inhibition of Yield
NOEC	3.26
LOEC	9.92
EyC10	5.40 (3.26 – 9.92)
EyC20	5.77 (3.26 – 9.92)
EyC50	7.10 (3.26 – 9.92)

Cell Densities

	Initial measured test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
9.92		1		5074		9391	7915	14906
		2		5074		11245	8735	7481
		3		5074		13602	7058	5988
		Mean		5074		11413	7903	9458
3.26		1		5074		19170	127885	882978
		2		5074		18657	127344	729392
		3		5074		18776	141100	795743
		Mean		5074		18868	132110	802704
0.974		1		5074		17116	91646	602272
		2		5074		16144	99460	599824
		3		5074		21310	119710	578033
		Mean		5074		18190	103605	593376
0.323		1		5074		16628	87816	641997
		2		5074		20478	76283	531317
		3		5074		18079	131387	587877
		Mean		5074		18395	98495	587064
0.106		1		5074		15254	80707	515889
		2		5074		15898	75533	536282
		3		5074		17784	99665	578701
		Mean		5074		16312	85302	543624
Control		1		5074		18538	79173	455381
		2		5074		16402	73470	461560
		3		5074		19826	88862	599549
		4		5074		17727	86135	466550
		5		5074		17747	76611	463257
		6		5074		17919	101695	510678
		Mean		5074		18027	84324	492829

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]		[%]
9.92	1		0.359	76.4		9832	98.0
	2		0.129	91.5		2407	99.5
	3		0.055	96.4		914	99.8
	Mean	(+)	0.181	88.1	(+)	4384	99.1
3.26	1		1.72	-12.9		877904	-80.0
	2		1.66	-8.68		724318	-48.5
	3		1.69	-10.6		790669	-62.1
	Mean	(-)	1.69	-10.7	(+)*	797630	-63.5
0.974	1		1.59	-4.50		597198	-22.4
	2		1.59	-4.41		594750	-21.9
	3		1.58	-3.60		572959	-17.5
	Mean	(-)	1.59	-4.17	(-)	588302	-20.6
0.323	1		1.61	-5.89		636923	-30.6
	2		1.55	-1.75		526243	-7.89
	3		1.58	-3.97		582803	-19.5
	Mean	(-)	1.58	-3.87	(-)	581990	-19.32
0.106	1		1.54	-1.11		510815	-4.73
	2		1.55	-1.96		531208	-8.91
	3		1.58	-3.62		573627	-17.6
	Mean	(-)	1.56	-2.23	(-)	538550	-10.4
Control	1		1.50			450307
	2		1.50			456486
	3		1.59			594475
	4		1.51			461476
	5		1.51			458183
	6		1.54			505604
	Mean		1.52			487755

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	1.30	1.45	1.75	1.50	0.231	15.4	17.5
	2	1.17	1.50	1.84	1.50	0.332	22.1
	3	1.36	1.50	1.91	1.59	0.284	17.9
	4	1.25	1.58	1.69	1.51	0.228	15.2
	5	1.25	1.46	1.80	1.51	0.276	18.4
	6	1.26	1.74	1.61	1.54	0.246	16.0
				Mean	1.52
				SD ±	0.04
				CV [%]	2.33

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study 1-Chlor-2,5-dimethoxy-4-nitrobenzol trocken was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) after 72 h were 6.46 (3.26 – 9.92) mg/L and 7.10 (3.26 – 9.92) mg/L, respectively. All effect levels are given based on initial measured concentrations of 1-Chlor-2,5-dimethoxy-4-nitrobenzol trocken.

Executive summary:

The toxicity of 1-Chlor-2,5-dimethoxy-4-nitrobenzol trocken (batch number DALA 053552) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from October 16 to 23, 2009 with the definitive exposure phase being conducted from October 20 to 23, 2009. The aim of the study was to assess the effects on growth rate and yield over a period of 72 h. Due to the poor solubility of the test substance the test solutions were prepared according to OECD (diffucult substances).

All effect values are given based on initial measured concentrations of the test item.

NOEC, LOEC and EC - values of 1-Chlor-2,5-dimethoxy-4-nitrobenzol trocken (0-72 h)

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

                               

	Rate-related Inhibition
NOEC	3.26
LOEC	9.92
ErC10	4.12 (3.26 – 9.92)
ErC20	4.60 (3.26 – 9.92)
ErC50	6.46 (3.26 – 9.92)
	Inhibition of Yield
NOEC	3.26
LOEC	9.92
EyC10	5.40 (3.26 – 9.92)
EyC20	5.77 (3.26 – 9.92)
EyC50	7.10 (3.26 – 9.92)