3-amino-2-chlor-6-methylphenol

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:

2016-10-10 to 2016-10-13

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-amino-2-chlor-6-methylphenol

EC Number:

283-144-9

EC Name:

3-amino-2-chlor-6-methylphenol

Cas Number:

84540-50-1

Molecular formula:

C7H8ClNO

IUPAC Name:

3-amino-2-chloro-6-methylphenol

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
The test concentrations of A094 5-Amino-6-chloro-o-cresol were analytically verified via HPLC-DAD at the start (0 hours) and at the end of the exposure (72 hours) with algae.

Test solutions

Vehicle:

no

Details on test solutions:

Stock solution, media preparation
100 mg test item/L, freshly prepared with dilution water. The dispersion was treated with ultrasound for 4 minutes at room temperature.

Test concentrations (nominal)
5 concentrations were tested in a geometrical series with a dilution factor of √10: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L. The dilution levels are based on the results of a preliminary range finding test (non-GLP).
 
Control
Six replicates (without test item) were incubated under the same conditions as the test concentrations.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

The method was modified according to OECD 23. The test item solutions are orange. Since this may affect the light intensities for algae cultures the volumes of test media in test vessels were lowered in order to minimize the water layer and path length.

Post exposure observation period:

No

Test conditions

Hardness:

Not specified

Test temperature:

min.: 21.0 max.: 22.5 mean value: 21.8

pH:

Nominal test item concentration Initial measured test item concentration pH-values
[mg/L] [mg/L] Start; 0 hours End; 72 hours
100 101 7.83 7.66
31.6 33.2 7.90 7.78
10.0 10.4 7.96 8.03
3.16 3.32 8.04 8.32
1.00 1.01 8.13 8.75
Control 8.03 8.91

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

Nominal: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L
initial measured test item concentration of: 1.01 – 3.32 – 10.4 – 33.2 – 101 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 25 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 25 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: Nominal: approximately 2 - 5 x 10^3 cells/mL, Actual: mean 6305 cells/mL
- Control end cells density: Mean 2004226 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2



OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, mean value: 5618 lux,



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. Self-fluorescence was observed in the range finding test. A separate replicate per test item concentration was prepared without algae and treated the same as the other test replicates. The self-fluorescence of each measuring point was taken into account in the evaluation.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.01 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

36.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 32.6 - 40.9

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.01 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.73 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 5.27 - 8.46

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

34.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 31.1 - 39.1

Effect concentrations 7

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

6.42 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: CI: 5.01 - 8.09

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
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA ALDRICH, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2016-10-11 to 2016-10-14 (with headspace). The reference item toxicity is in the valid range following test facility SOPS.

EC50-Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.436 0.764 ± 0.513
95% confidence interval 0.414 – 0.458
Yield inhibition
EyC50 0.230 0.402 ± 0.293
95% confidence interval 0.179 – 0.322
SD = Standard deviation

Reported statistics and error estimates:

EC-values and statistical analyses
EC10-, EC20- and EC,50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

NOEC, LOEC and statistical analyses
The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and DUNNETT’s test were 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 tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

 
Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
•Excel, MICROSOFT CORPORATION
•SigmaPlot, SPSS INC.
•GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Any other information on results incl. tables

Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities. All effect values are given based on the nominal test item concentrations.

 

 Cell Densities

Nominal test item concentration		Initial measured test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		[mg/L]		No.		0 hours		24 hours		48 hours		72 hours
100		101		1		6305		10550		9017		5775
			2		6305		14212		12132		5919
			3		6305		12084		11086		7365
			Mean		6305		12282		10745		6353
31.6		33.2		1		6305		18971		52765		125670
			2		6305		17299		67787		127153
			3		6305		19450		67835		155309
			Mean		6305		18573		62796		136044
10.0		10.4		1		6305		25745		158018		840458
			2		6305		19613		119888		470380
			3		6305		25401		144442		869832
			Mean		6305		23586		140783		726890
3.16		3.32		1		6305		23532		189190		1537174
			2		6305		22122		150051		1207971
			3		6305		24874		169176		1646495
			Mean		6305		23509		169472		1463880
1.00		1.01		1		6305		23509		178636		1917683
			2		6305		22320		172581		1824318
			3		6305		28389		183355		2100861
			Mean		6305		24739		178191		1947621
Control			1		6305		21198		163447		1952413
			2		6305		22923		158982		1851042
			3		6305		23476		196960		1990751
			4		6305		22644		186242		2098832
			5		6305		23780		177424		2119636
			6		6305		23698		183062		2012683
			Mean		6305		22953		177686		2004226

 

 Evaluation after 72 hours

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

 

Nominal test item concentration	Initial measured test item concentration	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[mg/L]	[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	101	1		-0.0293	100		-530	100
		2		-0.0211	100		-386	100
		3		0.0518	97		1060	100
		Mean	(+)	0.0005	99	(+)	48	100
31.6	33.2	1		1.00	48		119365	94
		2		1.00	48		120848	94
		3		1.07	44		149004	93
		Mean	(+)	1.02	47	(+)	129739	94
10.0	10.4	1		1.63	15		834153	58
		2		1.44	25		464075	77
		3		1.64	14		863527	57
		Mean	(+)	1.57	18	(+)	720585	64
3.16	3.32	1		1.83	5		1530869	23
		2		1.75	9		1201666	40
		3		1.86	3		1640190	18
		Mean	(+)	1.81	6	(+)	1457575	27
1.00	1.01	1		1.91	1		1911378	4
		2		1.89	2		1818013	9
		3		1.94	-1		2094556	-5
		Mean	(-)	1.91	1	(-)	1941316	3
Control		1		1.91			1946108
		2		1.89			1844737
		3		1.92			1984446
		4		1.94			2092527
		5		1.94			2113331
		6		1.92			2006378
		Mean		1.92			1997921

 

Section-by-Section and Average Specific Growth Ratesof the Control Group (0 - 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.21	2.04	2.48	1.91	0.644	33.7	30.2
	2	1.29	1.94	2.45	1.89	0.583	30.8
	3	1.31	2.13	2.31	1.92	0.531	27.7
	4	1.28	2.11	2.42	1.94	0.591	30.5
	5	1.33	2.01	2.48	1.94	0.580	29.9
	6	1.32	2.04	2.40	1.92	0.547	28.5
				Mean	1.92
				SD ±	0.02
				CV [%]	0.86

 

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, A094 5-Amino-6-chloro-o-cresol was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 34.6 (31.1 – 39.1) mg/L and 6.42 (5.01 – 8.09) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were >1.00 mg/L (based on supporting information, see Appendix), respectively.
In this study, A094 5-Amino-6-chloro-o-cresol was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on initial measured test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 36.3 (32.6 – 40.9) mg/L and 6.73 (5.27 – 8.46) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 1.01 mg/L, respectively.

Executive summary:

The toxicity of A094 5-Amino-6-chloro-o-cresol (Batch no.:20151025) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 C.3from 2016-09-05 to 2016-10-13, with the definitive exposure phase from 2016-10-10 to 2016-10-13 at the test facility.The aim of the study was the determination of NOEC, LOEC, EC₁₀, EC₂₀- and EC₅₀-values of growth rate and yield over a period of 72 hours.

A 1^st Definitive Study was conducted but results were insufficient to evaluate EC₅₀-values.

The 2^ndstudy was conducted under static conditions with an initial cell density of 6305 cells/mL. A stock solution of 100 mg test item/L was freshly prepared with dilution water and treated with ultrasound for 4 minutes at room temperature. The definitive test was carried with five concentrations in a geometrical series with a dilution factor of √10:1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L, corresponding to the initial measured test item concentration of: 1.01 – 3.32 – 10.4 – 33.2 – 101 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

The test media were clear and colorless at the start of the exposure. A concentration related color-change was observed throughout the test period fromcolorlessto clear orange. The concentrations of the test item in all test concentrations and the control were analytically verified by HPLC-DAD at the start and the end of exposure.
The measured concentrations of the test item in the fresh media (0 h) were between 101 and 105% of the nominal values. The measured concentrations in the old media (72 h) were between 80 an 89% of the nominal values.

Therefore, all effect values given are based on the nominal and initial measured test item concentrations ofthe test item.

 

NOEC, LOEC, EC_x-values and 95% Confidence Intervals of the test item (0 - 72 hours)

	Inhibition ofGrowth Rate
	Nominal test item concentration [mg/L]	Initial measured test item concentration [mg/L]
NOEC	>1.00*	1.01
LOEC	3.16	3.32
ErC10	5.34 (3.97 – 7.11)	5.54 (4.11 – 7.39)
ErC20	11.1 (9.03 – 13.2)	11.6 (9.45 – 13.8)
ErC50	34.6 (31.1 – 39.1)	36.3 (32.6 – 40.9)
	Inhibition ofYield
NOEC	>1.00*	1.01
LOEC	3.16	3.32
EyC10	1.60 (<1.00 – 2.37)	1.66 (< 1.01 – 2.47)
EyC20	2.52 (1.81 – 3.48)	2.64 (1.88 – 3.64)
EyC50	6.42 (5.01 – 8.09)	6.73 (5.27 – 8.46)

* = Based on supporting information from the 1^stDefinitive Study, the NOEC is considered to be higher than 1.00 mg/L.