2'-chloroacetoacetanilide

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:

2009-08-31 to 2009-09-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted and reported according to the OECD Guideline for the testing of chemicals 201 and principles of GLP.

Cross-reference

Reason / purpose for cross-reference:

reference to same 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

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2'-chloroacetoacetanilide

EC Number:

202-269-1

EC Name:

2'-chloroacetoacetanilide

Cas Number:

93-70-9

Molecular formula:

C10H10ClNO2

IUPAC Name:

2'-chloroacetoacetanilide

Details on test material:

- Molecular formula: C10H10ClNO2
- Molecular weight: 211.65 g/mol
- Physical state: Solid, white powder
- Expiration date of the lot/batch: 2009-12-19
- Storage condition of test material: Room temperature, protected from moisture and light

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No details available.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The limit concentration and the control were analytically verified at the beginning and the end of the test via HPLC-DAD analysis.
- Sampling method: 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 and treated with ultrasound for 10 min at room temperature.
- Eluate: Dilution water
- Differential loading: 100 mg/L (nominal) corresponding to a geometric mean measured test item concentration of 71.1 mg/L
- 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

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:

Geometric mean measured test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
71.1 7.83 8.39
Control 7.92 8.71

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 1061121 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)

Nominal
Test Item Concentration
[mg/L] Rate-related inhibition[%] Yield Inhibition[%]
100 -6 -41
10 -4 -22
1 -1 -7

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

71.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield inhibition

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 71.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield inhibition

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 71.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield inhibition

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:

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 Acetessig-o-chloranilid TTR
(0-72 h)

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

                               

	Rate-related Inhibition
NOEC	71.1
LOEC	> 71.1
ErC10	> 71.1
ErC20	> 71.1
ErC50	> 71.1
	Inhibition of Yield
NOEC	71.1
LOEC	> 71.1
EyC10	> 71.1
EyC20	> 71.1
EyC50	> 71.1

Cell Densities

	Geometric mean measured test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 h		24 h	48 h	72 h
71.1		1		5074		50490	324042	1553857
		2		5074		45812	289835	1314171
		3		5074		44122	278118	1403617
		4		5074		54795	369429	1546654
		5		5074		46697	345329	1665369
		6		5074		45881	360396	1716652
		Mean		5074		47966	327858	1533387
Control		1		5074		53089	335924	1077269
		2		5074		39494	226347	902601
		3		5074		36644	315907	1228621
		4		5074		43999	294452	892806
		5		5074		46705	251480	1048409
		6		5074		46177	327334	1217018
		Mean		5074		44351	291907	1061121

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]		[%]
71.1	1		1.908	-7.30		1548783	-46.66
	2		1.852	-4.16		1309097	-23.96
	3		1.874	-5.39		1398543	-32.43
	4		1.907	-7.21		1541580	-45.98
	5		1.931	-8.60		1660295	-57.22
	6		1.941	-9.17		1711578	-62.07
	Mean		1.902	-6.97		1528313	-44.72
Control	1		1.786			1072195
	2		1.727			897527
	3		1.830			1223547
	4		1.723			887732
	5		1.777			1043335
	6		1.827			1211944
	Mean		1.778			1056047

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	2.348	1.845	1.165	1.786	0.5935	33.3	25.9
	2	2.052	1.746	1.383	1.727	0.3348	19.4
	3	1.977	2.154	1.358	1.830	0.4179	22.8
	4	2.160	1.901	1.109	1.723	0.5474	31.8
	5	2.220	1.684	1.428	1.777	0.4042	22.7
	6	2.208	1.958	1.313	1.827	0.4619	25.3
				Mean	1.778
				SD ±	0.05
				CV [%]	2.60

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study Acetessig-o-chloranilid TTR was found not to inhibit the growth rate of the freshwater green alga Desmodesmus subspicatus after 72 h at 71.1 mg/L. Acetessig-o-chloranilid TTR caused a stimulation of alga growth. For details of effect values please refer to Table 1. All effect levels are given based on geometric mean measured concentrations of Acetessig-o-chloranilid TTR.

Executive summary:

The study was conducted and reported 2009 according to the OECD Guideline for the testing of chemicals 201 and principles of GLP.

The toxicity of Acetessig-o-chloranilid TTR to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201. 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. Based on a preliminary test, a limit test with nominal concentration level of 100 mg/L 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 Acetessig-o-chloranilid TTR were analysed at limit concentration level at test start and test end via HPLC-DAD analysis. The measured recovery rates of the test item at test start and end were 98 % and 52 % of the nominal value, respectively. All effect values are given based on geometric mean measured concentration of the test item. Acetessig-o-chloranilid TTR caused a distinctive stimulation of growth.

NOEC, LOEC, EC - values and 95 % Confidence Intervals of Acetessig-o-chloranilid TTR
(0-72 h)

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

                               

	Rate-related Inhibition
NOEC	71.1
LOEC	> 71.1
ErC10	> 71.1
ErC20	> 71.1
ErC50	> 71.1
	Inhibition of Yield
NOEC	71.1
LOEC	> 71.1
EyC10	> 71.1
EyC20	> 71.1
EyC50	> 71.1