4-chloro-2,5-dimethoxyaniline

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:

2009-08-18 to 2009-08-21

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:

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

Remarks:

GLP certificate and GLP compliance statement attached to full study report.

Test material

Test material information

Constituent 1

Reference substance name:

4-chloro-2,5-dimethoxyaniline

EC Number:

228-782-0

EC Name:

4-chloro-2,5-dimethoxyaniline

Cas Number:

6358-64-1

Molecular formula:

C8H10ClNO2

IUPAC Name:

4-chloro-2,5-dimethoxyaniline

Constituent 2

Reference substance name:

4-chloro-2,5-dimethoxyanilin

IUPAC Name:

4-chloro-2,5-dimethoxyanilin

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1 - 3.2 - 10 - 32 - 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 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: 160 mg/L, freshly prepared with dilution water, agitation
- Eluate: Dilution water
- Differential loading: 1.00 - 3.20 - 10.0 - 32.0 - 100 mg/L (factor √10)
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None

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: 22 °C, Min: 21 °C, Max: 23 °C

pH:

Geometric mean measured test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
88.5 7.44 6.94
26.0 7.59 7.00
7.91 7.70 7.35
2.55 7.84 7.50
0.785 8.05 7.87
Control 8.16 8.44

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Concentrations and Recovery Rates of 4-Chlor-2,5-dimethoxyanilin

Sampling date 2009-08-18
Test start 2009-08-21
Test end
Start of analysis 2009-08-18
2009-08-21

4-Chlor-2.5-dimethoxyanilin

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

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

[%] Geometric mean test item concentration
[mg/L]
100 90.8 91 86.3 86 88.5
32.0 28.2 88 23.9 75 26.0
10.0 9.56 96 6.55 66 7.91
3.20 3.13 98 2.08 65 2.55
1.00 0.977 98 0.631 63 0.785
Control < LOQ < LOQ < LOQ
The concentrations of 4-Chlor-2,5-dimethoxyanilin were measured after 0 and 72 h. The measured concentrations at test start well met the nominal ranges. Measured concentrations at the end of the test show a decrease of the test item. Therefore all effect values are given based on geometric mean measured concentrations.

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 852463 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 12 45
1 2 7

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.45 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: (3.89 - 7.90)

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: 0.695 (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 %. 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 4-Chlor-2,5-dimethoxyanilin
(0-72 h)

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

                               

	Rate-related Inhibition
NOEC	2.55
LOEC	7.91
ErC10	6.19 ( 0.785 - 10.6)
ErC20	9.91 (7.04 - 13.7)
ErC50	18.3 (14.5 - 21.7)
	Inhibition of Yield
NOEC	0.785
LOEC	2.55
EyC10	0.794 ( 0.785 - 1.44)
EyC20	1.53 ( 0.785 - 2.29)
EyC50	5.45 (3.89 - 7.90)

Cell Densities

	Geometric mean measured test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
88.5		1		5074		1060	0	0
		2		5074		0	0	0
		3		5074		917	0	0
		Mean		5074		659	0	0
26.0		1		5074		14639	23967	9707
		2		5074		20256	47378	43909
		3		5074		17985	47423	28026
		Mean		5074		17627	39589	27214
7.91		1		5074		21089	97468	386419
		2		5074		22913	111170	402253
		3		5074		22097	113339	485680
		Mean		5074		22033	107326	424784
2.55		1		5074		23454	120288	556815
		2		5074		28235	142473	557356
		3		5074		28268	125151	501572
		Mean		5074		26652	129304	538581
0.785		1		5074		23171	141137	819314
		2		5074		22056	166811	670853
		3		5074		27440	171600	870199
		Mean		5074		24222	159849	786789
Control		1		5074		29239	179869	920559
		2		5074		23680	157791	839781
		3		5074		28665	145110	869100
		4		5074		27259	143224	670865
		5		5074		22171	144626	813590
		6		5074		26095	168808	1000882
		Mean		5074		26185	156571	852463

Evaluation after 72 h

                   Statistically significant differences of growth rates and yield compared to
                   control values are marked (+), not significant differences are marked (-).          

                               

Geometric mean measured test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
88.5	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
26.0	1		0.216	87.32		4633	99.45
	2		0.719	57.82		38835	95.42
	3		0.570	66.60		22952	97.29
	Mean	(+)	0.502	70.58	(+)	22140	97.39
7.91	1		1.444	15.32		381345	55.00
	2		1.458	14.53		397179	53.13
	3		1.520	10.85		480606	43.28
	Mean	(+)	1.474	13.57	(+)	419710	50.47
2.55	1		1.566	8.18		551741	34.89
	2		1.566	8.16		552282	34.83
	3		1.531	10.22		496498	41.41
	Mean	(-)	1.555	8.85	(+)	533507	37.04
0.785	1		1.695	0.63		814240	3.91
	2		1.628	4.54		665779	21.43
	3		1.715	-0.55		865125	-2.09
	Mean	(-)	1.679	1.54	(-)	781715	7.75
Control	1		1.734			915485
	2		1.703			834707
	3		1.714			864026
	4		1.628			665791
	5		1.692			808516
	6		1.762			995808
	Mean		1.706			847389

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	1.751	1.817	1.633	1.734	0.093	5.38	7.33
	2	1.541	1.897	1.672	1.703	0.180	10.6
	3	1.732	1.622	1.790	1.714	0.085	4.98
	4	1.681	1.659	1.544	1.628	0.074	4.52
	5	1.475	1.875	1.727	1.692	0.203	12.0
	6	1.638	1.867	1.780	1.762	0.116	6.57
				Mean	1.706
				SD ±	0.05
				CV [%]	2.64

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see "Overall remarks"

Conclusions:

In this study 4-Chlor-2,5-dimethoxyanilin 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 specific growth rate (ErC50) and yield (EyC50) after 72 h were 18.3 (14.5 – 21.7) mg/L and 5.45 (3.89 – 7.90) mg/L, respectively.
All effect levels are given based on geometric mean measured concentrations of 4-Chlor-2,5-dimethoxyanilin.

Executive summary:

The toxicity of 4-Chlor-2,5-dimethoxyanilin(batch number DALC 011945) 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 , from August 18 to 21, 2009. 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 √10, nominal: 1.00 - 3.20 - 10.0 - 32.0 - 100 mg/L, corresponding to geometric mean measured concentrations of 0.785 - 2.55 - 7.91 - 26.0 - 88.5 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 4-Chlor-2,5-dimethoxyanilinwere 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 88 - 98 % of the nominal values. At the end of the test the active ingredient gave recoveries of 63 - 86 % of the nominal values. Therefore all effect values are given based on geometric mean measured concentrations of the test item.

NOEC, LOEC, EC - values and 95 % Confidence Intervals of 4-Chlor-2,5-dimethoxyanilin
(0-72 h)

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

                               

	Rate-related Inhibition
NOEC	2.55
LOEC	7.91
ErC10	6.19 ( 0.785 - 10.6)
ErC20	9.91 (7.04 - 13.7)
ErC50	18.3 (14.5 - 21.7)
	Inhibition of Yield
NOEC	0.785
LOEC	2.55
EyC10	0.794 ( 0.785 - 1.44)
EyC20	1.53 ( 0.785 - 2.29)
EyC50	5.45 (3.89 - 7.90)