2-[[4-[ethyl(2-hydroxyethyl)amino]phenyl]azo]-6-methoxy-3-methylbenzothiazolium chloride

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05.08.-05.09.1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Official Journal of the European Communities, L 133, 30/05/1988

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Composite samples (about 150 ml per concentration and control) of each test concentration were drawn by mixing identical volumes of the test solutions taken from the approximate center of the test vessels. They were taken immediately before exposure and after 72 hours exposure and kept at -18 °C to -22 °C until analysis.

Vehicle:

no

Details on test solutions:

Test concentrations were prepared from stock solution (20 mg test substance mixed and made up to 5000 ml test water).
Calculated amounts of the stock solution to produce the desired test concentrations were given into the water and were homogeneously distributed. The algae were then transfered into the flasks.
The test substance was visually homogeneously distributed in the test vessels at all test times and concentrations.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: Scenedesmus subspicatus
- Source: CIBA-GEIGY Ltd., testing facility
- Method of cultivation: according to guideline

ACCLIMATION
- Acclimation period: 3 days preculture at test conditions
- Culturing media and conditions (same as test or not): same
- Any deformed or abnormal cells observed: none reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

no data

Test temperature:

23 °C +/- 1 °C

pH:

7.8-7.9

Dissolved oxygen:

no data

Salinity:

n.a.

Conductivity:

no data

Nominal and measured concentrations:

Nominal: 0 (blank), 0.000123, 0.00037, 0.0011, 0.0033, 0.01 mg/L test substance

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks on lab shaker
- Type: open
- Size, fill volume: 100 ml, 50 ml
- Aeration: no
- Initial cell density: 10500 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: composition according to guideline


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: cold white fluorescent light, 119 µE/m² sec (mean, spherical unit)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell densities: TOA cell counter at 24, 48, and 72 h

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.008 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 95 % confidence limit

Remarks:

0.0071-0.0081 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.003 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

>= 0.01 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: calculated from raw data in study report

Details on results:

- Lower specific growth rate in control cultures on day one compared to the total average specific growth rate indicates a lag phase.
- It was demonstrated that a strong adsorption of the test substance occurs on polar surfaces. The proportion of test substance adhering on the glass walls and on the algae is not known. Analytical results seem to indicate an elimination of the test substance from the aqueous phase into the algae. Due to this situation the data are based and calculated on nominal values.
- Any stimulation of growth found in any treatment: yes

Reported statistics and error estimates:

The EbC-50 values were calculated according to BERKSON, JASA 48(1953),569-599.
The NOEbC value is calculated according to the modified DUNNETT'S MULTIPLE COMPARISON TEST(MARCUS and al., BIOMETRIKA 63, 655-660, 1976).

Nominal concentration [mg/L]		Cell densities cells/ml * 10000
		0 h	24 h	48 h	72 h
Control	R1	1.05	1.00	5.00	31.50
	R2	1.05	0.70	4.00	15.50
	R3	1.05	0.60	3.50	23.50
	R4	1.05	0.50	2.90	24.00
	R5	1.05	0.60	2.80	26.00
	R6	1.05	1.30	4.50	52.00
	mean	1.05	0.78	3.78	28.75
0.000123	R1	1.05	0.80	5.80	49.50
	R2	1.05	1.00	3.90	32.00
	R3	1.05	0.70	4.70	48.50
	mean	1.05	0.83	4.80	43.33
0.00037	R1	1.05	0.70	3.30	23.50
	R2	1.05	0.50	3.80	37.50
	R3	1.05	0.90	4.10	31.00
	mean	1.05	0.70	3.73	30.67
0.0011	R1	1.05	0.60	7.90	107.00
	R2	1.05	1.00	8.50	37.50
	R3	1.05	0.90	6.80	50.50
	mean	1.05	0.83	7.73	65.00
0.0033	R1	1.05	1.00	6.10	30.50
	R2	1.05	1.00	4.60	36.00
	R3	1.05	1.20	5.20	48.00
	mean	1.05	1.07	5.30	38.17
0.01	R1	1.05	0.80	0.90	3.00
	R2	1.05	1.00	0.50	2.50
	R3	1.05	0.60	1.20	11.50
	mean	1.05	0.80	0.87	5.67

Multiplication factor N 72h/ N 0h: 27

Inhibition

Nominal conc. [mg/L]	Areas under Growth Curves (A) * 10000						Mean A	Inhibition* IA 0-72 h [%]
	1	2	3	4	5	6
Blank	457	235	316	305	331	698	390	0.0
0.000123	688	439	647					0.0
0.00037	314	489	428					0.0
0.0011	1425	614	725					0.0
0.0033	471	501	665					0.0
0.01	13	3	117					88.7

* Promotion of algal growth compared to control is indicated as 0

Analytical determination of test concentrations

Nominal conc. [mg/L]	Measured concentrations
	Initial (0 h)		End (72 h)
	mg/L	%	mg/L	%
Blank	< 0.0005	-	< 0.0005	-
0.000123	nd	-	nd	-
0.00037	nd	-	nd	-
0.0011	0.0007	64	< 0.0005	< 45
0.0033	0.002	61	< 0.0005	< 45
0.01	0.007	70	0.001	10

nd: not determined (below the detection limit).

Recovery rate: 67.7 % (n=4, S_{rel .}= 6.9 % ) in the range of the nominal concentrations from 0.0033 to 0.01 mg/L.

This recovery was taken into account for calculation of all measured test concentrations.

Inhibition of growth rate (calculated from raw data)

Nominal concentration [mg/L]		Cell densities cells/ml * 10000				Section-by-section growth rate per day			Average specific growth rate µ	%Ir
		0 h	24 h	48 h	72 h	0-1 d	1-2 d	2-3 d	0-72 h	0-72 d
Control	R1	1.05	1.00	5.00	31.50	-0.002	0.1	0.1	0.05
	R2	1.05	0.70	4.00	15.50	-0.4	1.7	1.4	0.04
	R3	1.05	0.60	3.50	23.50	-0.6	1.8	1.9	0.04
	R4	1.05	0.50	2.90	24.00	-0.7	1.8	2.1	0.04	/
	R5	1.05	0.60	2.80	26.00	-0.6	1.5	2.2	0.04
	R6	1.05	1.30	4.50	52.00	0.2	1.2	2.4	0.05
	mean	1.05	0.78	3.78	28.75	-0.3	1.4	1.7	0.05
0.000123	R1	1.05	0.80	5.80	49.50	-0.3	2.0	2.1
	R2	1.05	1.00	3.90	32.00	0.0	1.4	2.1
	R3	1.05	0.70	4.70	48.50	-0.4	1.9	2.3
	mean	1.05	0.83	4.80	43.33	-0.2	1.7	2.2	0.05	-14.80
0.00037	R1	1.05	0.70	3.30	23.50	-0.4	1.6	2.0
	R2	1.05	0.50	3.80	37.50	-0.7	2.0	2.3
	R3	1.05	0.90	4.10	31.00	-0.2	1.5	2.0
	mean	1.05	0.70	3.73	30.67	-0.4	1.7	2.1	0.05	-4.13
0.0011	R1	1.05	0.60	7.90	107.00	-0.6	2.6	2.6
	R2	1.05	1.00	8.50	37.50	0.0	2.1	1.5
	R3	1.05	0.90	6.80	50.50	-0.2	2.0	2.0
	mean	1.05	0.83	7.73	65.00	-0.3	2.2	2.0	0.06	-27.32
0.0033	R1	1.05	1.00	6.10	30.50	0.0	1.8	1.6
	R2	1.05	1.00	4.60	36.00	0.0	1.5	2.1
	R3	1.05	1.20	5.20	48.00	0.1	1.5	2.2
	mean	1.05	1.07	5.30	38.17	0.0	1.6	2.0	0.05	-10.89
0.01	R1	1.05	0.80	0.90	3.00	-0.3	0.1	1.2
	R2	1.05	1.00	0.50	2.50	0.0	-0.7	1.6
	R3	1.05	0.60	1.20	11.50	-0.6	0.7	2.3
	mean	1.05	0.80	0.87	5.67	-0.3	0.0	1.7	0.02	47.98

Validity criteria fulfilled:

yes

Remarks:

validity criteria as laid down in the Official Journal of the European Communities No L 133 from 30/05/1988

Conclusions:

The 72-h EbC50 and NOEbC of the test item to the green algae (Scendesmus subspicatus) is 0.0077 mg/L and 0.0033 mg/L, respectively.
The 72-h ErC50 was calculated from the raw data given in the study report: > 0.01 mg/L.

Executive summary:

In a 72 hour toxicity study, the cultures of Scenedesmus subspicatus were exposed to the test item at nominal concentrations of 0 (blank), 0.000123, 0.00037, 0.0011, 0.0033 and 0.01 mg/L under static conditions in accordance with the Official Journal of the European Communities No L 133 from 30/05/1988. The NOEC and EC₅₀ values based on cell density were 0.0033 and 0.077 mg/L, respectively.  The % growth inhibition (areas under growth curves) in the treated algal culture as compared to the control ranged from 0 (stimulation of growth) to 88.7% at the highest concentration tested.

Based on the raw data given in the study report, the ErC value for inhibition of the growth rate was calculated. At the highest concentration tested, the inhibition of the growth rate was 48 %.

 

The following abnormalities were noted: 

- Lower specific growth rate in control cultures on day one compared to the total average specific growth rate indicates a lag phase.

- It was demonstrated that a strong adsorption of the test substance occurs on polar surfaces. The proportion of test substance adhering on the glass walls and on the algae is not known. Analytical results seem to indicate an elimination of the test substance from the aqueous phase into the algae. Due to this situation the data are based and calculated on nominal values.

- All test concentrations except for the highest substance concentration of 0.01 mg/L resulted in promotion of algal growth compared to the control cultures.

 

This toxicity study is classified as supplementary and satisfies the guideline requirements for an Algal Inhibition Test according to the Official Journal of the European Communities No L 133 30/05/1988.

 

Results Synopsis

Test Organism: Scenedesmus subspicatus

Test Type: Static

 

72 hr EbC₅₀: 0.0077 mg/L                95% C.I.:  0.0071-0.0081 mg/L

72 hr NOEbC:  0.0033 mg/L                 

Endpoint(s) Effected:  cell number

72 hr ErC₅₀ > 0.01 mg/L                           

Endpoint(s) Effected:  growth rate

Description of key information

In a 72 hour toxicity study, green algae were exposed to the test item at five different concentrations of up to 0.01 mg/L (nominal).

The NOEC and EC₅₀values based on cell density were 0.0033 and 0.077 mg/L, respectively.  The % growth inhibition (areas under growth curves) in the treated algal culture as compared to the control ranged from 0 (stimulation of growth) to 88.7% at the highest concentration tested.

Based on the raw data given in the study report, the ErC₅₀ value for inhibition of the growth rate was calculated. At the highest concentration tested, the inhibition of the growth rate was 48 %.

 

Based on a worst case approach the ErC₅₀ is considered to be 0.01 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.01 mg/L