1-chloro-2,5-dimethoxybenzene

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:

2009-07-14 to 2009-07-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to current OECD-Guidelines and 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:

1-chloro-2,5-dimethoxybenzene

EC Number:

218-267-9

EC Name:

1-chloro-2,5-dimethoxybenzene

Cas Number:

2100-42-7

Molecular formula:

C8H9ClO2

IUPAC Name:

2-chloro-1,4-dimethoxybenzene

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.00 - 3.20 - 10.0 - 32.0 - 100 mg/L
- Sampling method: All test concentrations and control were analytically verified at the beginning and the end of the test via HPLC-DAD 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 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 200 mg/L was freshly prepared with dilution water. Dispersion treatment was ultrasonification for 15 min at room temperature.
- Eluate: Dilution water
- Differential loading: 1,00 - 3.20 - 10.0 - 32.0 - 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): 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

Test conditions

Hardness:

Not measured

Test temperature:

Mean: 23 °C, Min: 22 °C, Max: 24 °C

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
100 7.44 6.80
32.0 7.52 6.78
10.0 7.58 7.65
3.20 7.69 7.69
1.00 7.71 7.73
Control 8.11 8.55

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Concentrations and Recovery Rates of Chlor-dimethylhydrochinon

Sampling date 2009-07-14
Test start 2009-07-17
Test end
Start of analysis 2009-07-14
2009-07-17

Chlor-dimethylhydrochinon
Nominal test item concentration
[mg/L] Meas.
conc.
[mg/L] RR

[%] Meas.
conc.
[mg/L] RR

[%]
100 97.8 98 88.3 88
32.0 28.9 90 28.4 89
10.0 9.56 96 9.17 92
3.20 3.04 95 2.62 82
1.00 0.941 94 0.822 82
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 1345978 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

[%]
100 100 100
10 17 57
1 0 -1

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: (8.49 - 9.67)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

13.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (12.9 - 13.7)

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 biomass integrals and growth rates.
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 %. These findings were marked biological not significant. Following the recommendations of the UBA (HEGER ET AL. 1998), inhibitions > 10 % are significant.

Any other information on results incl. tables

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

                   based on nominal test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	3.20
LOEC	10.0
ErC10	8.93 (8.64 - 9.19)
ErC20	10.3 (9.94 - 10.5)
ErC50	13.3 (12.9 - 13.7)
	Inhibition of Yield
NOEC	3.20
LOEC	10.0
EyC10	6.73 (6.27 - 7.21)
EyC20	7.35 (6.90 - 7.85)
EyC50	9.10 (8.49 - 9.67)

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		0	0	0
		2		5074		0	0	0
		3		5074		0	0	0
		Mean		5074		0	0	0
32.0		1		5074		2717	761	359
		2		5074		4459	2417	1564
		3		5074		4881	3705	2721
		Mean		5074		4019	2294	1548
10.0		1		5074		41785	99915	439908
		2		5074		37513	137455	517353
		3		5074		38177	146085	543859
		Mean		5074		39158	127818	500373
3.20		1		5074		43110	358269	1603102
		2		5074		46824	393266	1745987
		3		5074		45693	376973	1535707
		Mean		5074		45209	376169	1628265
1.00		1		5074		41646	379236	1612741
		2		5074		41076	340536	1429701
		3		5074		43286	316190	1470078
		Mean		5074		42003	345321	1504173
Control		1		5074		44200	332188	1458721
		2		5074		50080	273095	1161020
		3		5074		42306	333882	1545083
		4		5074		49469	345005	1465863
		5		5074		42815	271886	1194558
		6		5074		39789	301787	1250621
		Mean		5074		44777	309641	1345978

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		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
32.0	1		-0.883	100.00		-4715	100.00
	2		-0.392	100.00		-3510	100.00
	3		-0.208	100.00		-2353	100.00
	Mean	(+)	-0.494	100.00	(+)	-3526	100.00
10.0	1		1.487	19.95		434834	67.57
	2		1.542	17.04		512279	61.80
	3		1.558	16.15		538785	59.82
	Mean	(+)	1.529	17.71	(+)	495299	63.06
3.20	1		1.919	-3.25		1598028	-19.18
	2		1.947	-4.78		1740913	-29.83
	3		1.904	-2.48		1530633	-14.15
	Mean	(-)	1.923	-3.50	(+)*	1623191	-21.05
1.00	1		1.921	-3.35		1607667	-19.89
	2		1.880	-1.19		1424627	-6.24
	3		1.890	-1.69		1465004	-9.25
	Mean	(-)	1.897	-2.08	(-)	1499099	-11.79
Control	1		1.887			1453647
	2		1.811			1155946
	3		1.906			1540009
	4		1.889			1460789
	5		1.820			1189484
	6		1.836			1245547
	Mean		1.858			1340904

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	2.165	2.017	1.480	1.887	0.3605	19.1	20.0
	2	2.289	1.696	1.447	1.811	0.4327	23.9
	3	2.121	2.066	1.532	1.906	0.3252	17.1
	4	2.277	1.942	1.447	1.889	0.4179	22.1
	5	2.133	1.848	1.480	1.820	0.3272	18.0
	6	2.059	2.026	1.422	1.836	0.3590	19.6
				Mean	1.858
				SD ±	0.04
				CV [%]	2.18

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study Chlor-dimethylhydrochinon was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 h with the following effect values (nominal concentrations): The EC50-values with 95 % confidence intervals for specific growth rate (ErC50) and yield (EyC50) after 72 h were 13.3 (12.9 – 13.7) mg/L and 9.10 (8.49 – 9.67) mg/L, respectively.
All effect levels are given based on nominal concentrations of Chlor-dimethylhydrochinon

Executive summary:

The toxicity of Chlor-dimethylhydrochinon(batch number KB 288-11.1.2008) 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 July 14 to 17, 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 10³cells/mL. Five concentration levels were tested in a geometrical series with a dilution factor of √10, nominal: 1.00 - 3.20 - 10.0 - 32.0 - 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 Chlor-dimethylhydrochinonwere analysed at all concentration levels at test start and test end via HPLC-DAD analysis. The measured concentrations at test start were in the range of 90 - 98 % of the nominal values. At the end of the test the active ingredient gave recoveries of 82 - 92 % of the nominal values. All effect values are given based on nominal concentrations of the test item.

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

                   based on nominal test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	3.20
LOEC	10.0
ErC10	8.93 (8.64 - 9.19)
ErC20	10.3 (9.94 - 10.5)
ErC50	13.3 (12.9 - 13.7)
	Inhibition of Yield
NOEC	3.20
LOEC	10.0
EyC10	6.73 (6.27 - 7.21)
EyC20	7.35 (6.90 - 7.85)
EyC50	9.10 (8.49 - 9.67)