2,2'-methylenebis[6-(1-methylcyclohexyl)-p-cresol]

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:

June 27 - 30, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD test guidelines in compliance with GLP and reported with a GLP certificate.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
None specified

Analytical monitoring:

yes

Details on sampling:

The determination of the test substance was performed via HPLC at the beginning and the end of the test.
Sampling was carried out directly from additional vessels at test start (0 (h), new media) and at the test end (72 (h), old media). Before sampling the test media were mixed.

Vehicle:

no

Details on test solutions:

5 (mL) inoculum + 5 (mL) solution of test substance with appropriate dilution
Control: 5 (mL) inoculum + 5 (mL) demineralised water

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST SYSTEM: Scenedesmus subspicatus CHODA T SAG 86.81
Reason for the selection of the test system: According to the guideline Scenedesmus subspicatus
CHODAT SAG 86.81 is suitable for this kind of study.
Origin: Collection of algae cultures; Pflanzenphysiologisches Institut der Universität, Nikolausberger Weg 18, 0-37073 Göttingen
Culturing: DR.U.NOACK-LABORATORIUM FÜR ANGEWANDTE BIOLOGIE, Richthofenstr. 29, 0-31137 Hildesheim
Method of cultivation: In sterile glass flasks (vol. 2 (L)) filed with 2 (L) culture medium at room temperature. Light intensity amounted 35-70 (μE/m2 * s1) for 24 (h) per day (fluorescent tubes,
Typ Osram L 18W/25 weiß universal).
Culture medium: Nutrient medium according to KUHL (1962, Deutsch. Bot. Ges., Beiträge zur Physiologie und Morphologie der Aigen, 157-166. Fischer-Verlag)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

Not specified in the study report.

Test temperature:

22.5 - 23.6 (°C). nominal range: 23 ± 2 (°C)

pH:

7.83 to 7.97

Dissolved oxygen:

Not specified in the study report.

Salinity:

Not applicable - freshwater study

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal concentrations

Details on test conditions:

Test substance: Lowinox WSP
Stock solutions: 0.2 - 0.64 - 2.0 - 6.4 - 20 - 64 - 200 - 640 - 2000 (mg/L)
Test concentrations: 0.1 - 0.32 - 1.0 - 3.2 - 10 - 32 - 100 - 320 - 1000 (mg/L)
Dispersion Treatment: 30 (min) ultrasound at 40 (°C)
Vehicle: None
CONTROL: Four replicates with test medium (without test substance) were tested under the same test conditions as the test substance.
Test method: Static procedure
Duration of test: 72 (h), June 27 - 30, 1995
Replicates: Four replicates per concentration level and control
Self fluorescence: None
Frequency and duration of application: 1 x over 72 (h) at test start
Test container: Plastic cuvettes (diameter 50 (mm)), transparent, with top, volume 20 (mL)
Test volume: 10 (mL)
Test medium: According to OECD 201
Inoculum: A four-day-old preculture was used. Incubation was performed in 500 (mL) Erlenmeyer flasks with nutrient medium according to OECD 201.
Initial cell density: 1.0 - 1.6.104 (N/mL)
Composition of test medium: 5 (mL) inoculum + 5 (mL) solution of test substance with appropriate dilution
Control: 5 (mL) inoculum + 5 (mL) demineralised water
Temperature: 22.5 - 23.6 (°C). Nominal range: 23 ± 2 (°C)
Mixing during the test: permanent with shaker (70 (rpm))
Light intensity: Deviating from the test guideline the light intensity amounted 35-70 (μE/m2 * s1) for 24 (h) per day (fluorescent tubes, Typ Osram L 18W /25 weiß universal).
Light regime: 24 (h/d) light
Kind and frequency of measurement: Cell density was measured via Chlorophyll a-fluorescence (Impulsfluorometer, Kleinfeld company), excitation at 435 (nm), emission at 685 (nm). The cell density was measured at the beginning of the test and every 24 (h).
The pH-value at the beginning of the test was measured out of a mixture of two additional replicates of each concentration and control. At the end it was measured from a mixture of all replicates. The temperature was measured continuously by a hygro-thermographe.
Equipment: pH-Meter, pH 537 (WTW Company), Hygro-thermographe (Klimatherm company); Chlorophyll-fluorometer (Kleinfeld company); Shaker, SM 25 A (Buhler company)

TEST DESCRIPTION
The test substance was identified on May 30, 1995. A homogenity and solubility test with water and acetone was conducted from June 1 to 5 1995. The homogeneity was observed after 0, 4, 24 and 96 (h).
A preliminary range finding test was conducted with 4 concentration levels (four replicates each) of the test substance ranging from 0.1 - 100 (mg/L) setup in a geometric series of factor 10 (NON-GLP-state). The purpose of the preliminary range finding test conducted from June 6 to June 9, 1995, was to obtain information on the concentration levels to be used in the main study.
Based on the results of the preliminary test, a main test was performed with 9 concentration levels ranging from 0.1 - 1000 (mg/LJ in a geometrical series with a √10 factor.
A control with test medium (without test substance) (four replicates) was tested under the same test, conditions as the test groups.
A four-day-old preculture incubated at study conditions was used for the main study.
Chlorophyll-fluorescence was determined at the beginning and after 24, 48 and 72 (h).

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass & growth rate

Details on results:

IDENTIFICATION AND SOLUBILITY
The identification of the test substance was checked. Agreement was ascertained to the sponsor with regard to aggregate state, consistency, colour, melting point, batch-number and name of the test substance.
The test substance was not soluble in water and water with acetone in a concentration of 1000 (mg/L). After 0 h the test was partially dispersed in all replicates.

RESULTS OF THE INLIFE-PART: MAIN TEST
The results of the main test showed that the algal biomass was inhibited < 50 (%) at the concentrations of 32-320 (mg/L). The growth rate was not inhibited at a concentration of 1000 (mg/L).
Therefore the NOEC values were laid down at 10 (mg/L) for biomass and 1000 (mg/L) for growth rate.
Microscopic evaluation of the cells at the start of the incubation revealed no morphological abnormalities.

Results with reference substance (positive control):

Reference substance not reported in this study.

Reported statistics and error estimates:

Out of the fluorescence values the mean fluorescence of the replicates of each test concentration and control were calculated.
The fluorescence values were related to cell density values.
Out of the cell density values the growth rate, the inhibition of biomass and the rate-related inhibition after 72 (h) were calculated.
ECo- (NOEC), EC,o- and EC6o-values: the EC-values were deduced from the dose-response-relationship. There was no mathematical calculation.
Calculations were performed using software Sigma Plot 5.0, Jandel Scientific (1992).

Preliminary test results: fluorescence values (4 replicates of each concentration)

Conc. mg/[L]	Chlorophyll fluorescence		Self fluor. 0 [h]
	0 [h]	72 [h]
100	157 / 142 166 / 145	2414 / 2484 2128 / 2194	0
10	153 / 158 161 / 155	2782 / 2568 2800 / 2334	-
1	155 / 149 157 / 161	3050 / 3368 3156 / 2904	-
0.1	155 / 148 161 / 138	3290 / 3220 23460 / 3350	-
Contr.	144 / 155 146 / 155	3404 / 3552 3828 / 3542	-

 

EC-values (0-72 [h]) based on nominal concentrations

Inhibition of biomass growth	[mg/L]
EbC0	10
EbC10	10<EC10<1000
EbC50	>1000
Growth rates related inhibition	[mg/L]
ErC0	1000
ErC10	>1000
ErC50	>1000

 

Cell density (nominal concentrations)

Serial No.	Concentration (nominal) [mg/L]	0 h	24 h	48 h	72 h
		[N/mL] [*10^3]	[N/mL] [*10^3]	[N/mL] [*10^3]	[N/mL] [*10^3]
1	Control	11	37	169	420
2	0.1	10	39	174	447
3	0.32	11	39	169	416
4	1.0	11	41	177	456
5	3.2	11	35	150	419
6	10	12	43	170	428
7	32	12	34	141	402
8	100	13	35	141	384
9	320	15	37	128	396
10	1000	16	50	187	604

 

Evaluation after 72 h (nominal concentrations)

Serial No.	Concentration (nominal) [mg/L]	Inhibition of Biomass [%]	Growth Rate	Rate-related Inhibition [%]
1	Control	---	1.21	---
2	0.1	-6.01	1.26	-4.24
3	0.32	-0.06	1.21	0.29
4	1.0	-8.25	1.25	-3.45
5	3.2	5.69	1.21	0.51
6	10	-2.44	1.20	1.07
7	32	10.86	1.17	3.20
8	100	13.61	1.13	6.93
9	320	15.75	1.10	9.66
10	1000	-28.84	1.21	-0.46

Negative inhibition >10 [%] = increase of growth

 

REMARK: A stimulation of biomass growth was found at the concentration level 1000 [mg/L].

Validity criteria fulfilled:

yes

Conclusions:

The results of the main test showed that the algal biomass was inhibited < 50 (%) at the concentrations of 32-320 (mg/L). The growth rate was not inhibited at a concentration of 1000 (mg/L).
Therefore the NOEC values were laid down at 10 (mg/L) for biomass and 1000 (mg/L) for growth rate.

Executive summary:

The growth inhibition of Lowinox WSP to the unicellular algal specie Scenedesmus subspicatus CHODA T was conducted according to OECD Guideline No. 201 from June 27 - 30, 1995. Therefore test duration was 72 (h).

Test facility was DR.U.NOACK-LABORATORIUM FÜR ANGEWANDTE BIOLOGIE in 31137, Hildesheim, FRG.

Test substance was the product Lowinox WSP as pure substance. Batch number of the tested sample was 5E049. No carrier for dissolving the test substance was used. The test substance was not soluble in water and water with acetone in a concentration of 1000 (mg/L).

The test substance was applied once at test begin. A static test was conducted with the nominal concentrations:

0.1 - 0.32 - 1.0 - 3.2 - 10 - 32 - 100 - 320 - 1000 (mg/L)

selected on the basis of a preliminary static test.

The initial cell density was 1.0 - 1.6 x 10⁴(N/mL).

The concentrations of the active ingredient were determined in the concentration levels between 3.2 and 1000 (mg/L) at test start and test end. The recovery rates at the beginning of the test were in the range of 25 - 89 (%). At the end of the test they were in a range of 35 - 236 (%) Lowinox WSP. All effect concentrations are based on nominal concentrations of the formulated product.

The results of the main test showed that the algal biomass was inhibited < 50 (%) at the concentrations of 32-320 (mg/L). The growth rate was not inhibited at a concentration of 1000 (mg/L).

Therefore the NOEC values were laid down at 10 (mg/L) for biomass and 1000 (mg/L) for growth rate.

The growth inhibition effects of Lowinox WSP, based on nominal concentrations are summarized in the following table:

 

EC-values (0-72 [h]) based on nominal concentrations

Inhibition of biomass growth	[mg/L]
EbC0	10
EbC10	10<EC10<1000
EbC50	>1000
Growth rates related inhibition	[mg/L]
ErC0	1000
ErC10	>1000
ErC50	>1000

 

Microscopic evaluation of the cells at the start of the incubation revealed no morphological abnormalities. The pH of the media containing different concentrations of the test substance remained constant during the test.

Water quality parameters of the control (pH), measured at 0 and 72 (h), and temperature measured continuously, were determined to be within the acceptable limits.

Description of key information

Key value determined in laboratory study according to OECD Guideline 201. 

Key value for chemical safety assessment

EC50 for freshwater algae:

1 000 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L

Additional information

Test substance was the product Lowinox WSP as pure substance. Batch number of the tested sample was 5E049. No carrier for dissolving the test substance was used. The test substance was not soluble in water and water with acetone in a concentration of 1000 (mg/L).

The test substance was applied once at test begin. A static test was conducted with the nominal concentrations:

0.1 - 0.32 - 1.0 - 3.2 - 10 - 32 - 100 - 320 - 1000 (mg/L)

selected on the basis of a preliminary static test.

The initial cell density was 1.0 - 1.6 • 10⁴(N/mL).

The concentrations of the active ingredient were determined in the concentration levels between 3.2 and 1000 (mg/L) at test start and test end. The recovery rates at the beginning of the test were in the range of 25 - 89 (%). At the end of the test they were in a range of 35 - 236 (%) Lowinox WSP. All effect concentrations are based on nominal concentrations of the formulated product.

 

The results of the main test showed that the algal biomass was inhibited < 50 (%) at the concentrations of 32-320 (mg/L). The growth rate was not inhibited at a concentration of 1000 (mg/L).

Therefore the NOEC values were laid down at 10 (mg/L) for biomass and 1000 (mg/L) for growth rate.

 

Microscopic evaluation of the cells at the start of the incubation revealed no morphological abnormalities. The pH of the media containing different concentrations of the test substance remained constant during the test.

Water quality parameters of the control (pH), measured at 0 and 72 (h), and temperature measured continuously, were determined to be within the acceptable limits.