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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 28 to 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 202 (Daphnia sp. Acute Immobilisation 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 concentrations was performed via HPLC at test start and after 48 (h).
All test concentrations were determined.

Vehicle:

no

Details on test solutions:

TEST SUBSTANCE: Lowinox WSP
Dispersion treatment: 30 (min) ultrasound at 40 (°C)
Carrier: No carrier was used.
Stock solution: 1000 (mg/L)
Test concentrations: 100 - 180 - 320 - 580 - 1000 (mg/L)
CONTROL: 4 (2*) controls tested under the same conditions as the test groups.
*) The information in brackets refer to the test of the reference substance

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST SYSTEM: Daphnia magna STRAUS
Reason for the selection of test system: Daphnia magna STRAUS belongs, in accordance with the study guideline, to the suitable Daphnia types and are bred in the test facility.
Origin: lnstitut für Wasser-, Boden- und Lufthygiene des Bundesgesundheitsamtes, Corrensplatz, 14195 Berlin, FRG
Breeder: Dr.U.Noack-Laboratorium, 31137 Hildesheim, FRG
Culture: In 2-3 (L) glass vessels with approximatively 1.8 (L) culture medium, by 21 (°C) (temperatures of 20-25 (°C) were tolerated), in an incubator, 10 (h) illumination, illumination strength 1.5-3.0 (μmol*m-2*s-1).
Culture medium: Tap water of local origin, prepared by filtration over active charcoal with 5 (mL) Tetra Aqua Safe per 10 (L) media.
Feeding: 5 x weekly with Scenedesmus subspicatus, 100 - 150 (mL) per glass sample with an algae cell density of approximatively 6 * 105 - 9 * 105 (N/mL).

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

No post exposure observation period specified.

Hardness:

Total hardness: 15 [°dH]

Test temperature:

23.3 [°C]

pH:

7.93

Dissolved oxygen:

8.2 [mg/L]

Salinity:

Not applicable - freshwater study

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal concentrations

Details on test conditions:

TEST METHOD: static
Test duration: 48 (h), June 28 to 30, 1995
Application: 1 x within 48 (h)
Test vessels: glass beakers, 50 (mL)
Volume of the study medium: 20 (mL)
Dilution water: according to EEC 84/449, Annex
Adaptation: The study organisms were acclimated to the dilution water 2 (h) before test start
Number of study organisms per concentration and control: 20 animals, divided into 4 (2*) parallel samples, each with 5 (10*) animals
Number of animals per test vessel: 5 (10*)
Number of parallel samples per concentration: 4 (2*)
Age of test organisms: at maximum 24 (h) old
Test temperature: 18 - 22 [°C] ± 1 [°C] constant
Illumination: diffuse light, illumination range 1.5 - 5 [μmol *m-2*s-1]
Photoperiod: 16/8 (h) light/dark cycle
*) The information in brackets refer to the test of the reference substance

TEST DESCRIPTION
The test substance was identified on May 30, 1995.
Solubility tests of the test substance were carried out from June 1 to 5, 1995. The homogeneity was reported after 0, 4, 24 and 96 (h).
2 to 24 (h) old daphnids were used for the range finding and main study. They had been obtained by removing the mother animals twice within 22 (h).
A preliminary range finding test was conducted with 4 concentration levels of the test article ranging from 0.1 to 100 (mg/L), set up in a geometric series with a factor of 10 (NON-GLP-state).
Based on the results of the preliminary test a main test was performed with 5 concentrations ranging from 100 - 1000 (mg/L) in a geometrical series with a 1.8 factor to enable the determination of 0 and 100 % immobilization after 24 and 48 hours. In the preliminary test and in the main study the immobilization of the daphnia was observed after 24 and 48 hours of exposure.
The daphnids were acclimated to the dilution water 2 hours before test start.
The stock solution of 1000 (mg/L) was prepared with dilution water.
The reference substance was tested in five concentrations within the concentration range 0.32 - 3.2 (mg/L).
20 daphnids divided into 4 (2*) groups with 5 (10*) animals per test vessel were used.
All test beakers were placed in an incubator (light/dark cycle 16/8) for 48 (h) at 21 ± 1 (°C).
Observations, measurements and evaluation were carried out as described below.
*) The information in brackets refer to the test of the reference substance

TYPE AND FREQUENCY OF MEASUREMENT AND OBSERVATIONS
Prior to test start pH-value, O2-saturation, conductivity, total hardness and temperature of the dilution water were measured.
At the beginning of the test the water parameters (pH, O2) were measured in one replicate per concentration and controls. After 48 (h) the water parameters were measured in all replicates per concentration and control.
The temperature was recorded throughout the test with a thermohygrograph.
Immobility was determined in all groups after 24 and 48 (h).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate p.a.

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

IDENTIFICATION AND SOLUBILITY
The identity of the test substance was checked. Agreement was ascertained according to the sponsor with regard to state, consistency, color, melting point, batch no. and name of the test substance. Data relating to the characterization of the test substance are documented in an analytical certificate provided by the sponsor.
The test substance was not soluble in dilution water and in dilution water with the carrier Acetone. A milky suspension was observed. A part of the test substance was soluble. Therefore the test was done without a carrier.

PRELIMINARY TEST RESULTS (NON-GLP-state)
The percentage immobility was determined in all groups and controls after 24 and 48 hours of exposure. The immobilization rates are given in table form (see Any other information).
After 48 (h) of exposure the total rate of immobilization of daphnia was 0 % at the nominal concentration 100 (mg/L). No biologically significant immobilization of daphnia was seen at the nominal concentration 100 (mg/L) and lower after 48 (h).

SUMMARY OF SUPPORTING CHEMICAL ANALYSIS
The recovery rates of the nominal concentrations are summarized in table form (see Any other information).
Freshly prepared solutions (= new media) showed recovery rates ranging from 39 to 85 (%).
The 48 h solutions showed recovery rates in the range from 73 to 109 (%).
The mean recovery rates were 63 (%) after 0 (h) and 89 (%) after 48 (h).
All results are given as nominal concentrations.

RESULTS OF THE INLIFE PART (MAIN STUDY)
The percentage immobility was determined in all groups and control after 24 and 48 h.
The immobilization rates in (%) during 24 and 48 h of the test are given in table form (see Any other information).
After 24 and 48 (h) of exposure the total rate of immobilisation of daphnia were 0 (%) at the highest nominal concentration of the product of 1000 (mg/L).
Therefore the EC0 values were:
24 (h) EC0: ≥ 1000 (mg/L)
48 (h) EC0: ≥ 1000 (mg/L)
There was no concentration resulting in 100 (%) incapability of swimming during the test:
24 (h) EC100: > 1000 (mg/L)
48 (h) EC100: > 1000 (mg/L)
Because there was no mortality during the test the EC50 values could not be determined:
24 h EC50: > 1000 (mg/L) P = 95 (%): not determined
48 h EC50: > 1000 (mg/LI P = 95 (%): not determined

WATER PARAMETERS
The water parameters (O2, pH) of test and control groups in one replicate at the beginning of the test and in all replicates after 48 (h) are listed in table form (see Any other information).
pH-value, oxygen, conductivity, total hardness and temperature of the dilution water were measured at the beginning of the test. The results are listed in table form (see Any other information).
During the test the temperature was constant 20 (°C).

Results with reference substance (positive control):

The percentage immobility for the reference substance was determined after 24 (h). The EC0 and EC100 values were determined directly from the test results. The EC50 was determined in a probability network through interpolation according to standard procedures.
24 (h) EC values of the reference substance:
24 (h) EC0: 0.58 (mg/L)
24 (h) EC50: 1.30 (mg/L)
24 (h) EC100: 3.20 (mg/l)
The EC50 value of reference substance potassium dichromate after 24(h) is within the prescribed concentration range of 0.9 - 1.9 (mg/L) of quality criteria according to DIN 38412 L11.

Reported statistics and error estimates:

A statistical determination of the 24 and 48 (h) EC50 values was not performed.

Immobilization rates of the preliminary test

Conc. [mg/L]	24 [h]		MV	48 [h]		MV
	Rep. 1	Rep. 2		Rep. 1	Rep. 2
100	0	0	0	0	0	0
10	0	0	0	0	0	0
1	0	0	0	0	0	0
0.1	0	0	0	0	0	0
Control	0	0	0	0	0	0

MV = mean value

 

Summary of chemical analysis:

Recovery rates of the nominal

concentrations in the new and old media

Nominal conc. [mg/L]	Recovery rates [%]
	Media
	New 0 [h]	Old 48 [h]
1000	64 66	102 103
580	39 40	107 109
320	58 58	77 79
180	82 85	82 82
100	70 70	73 74
Mean	63	89
Control	n.d.	n.d.

n.d. = <determination limit

 

Number of daphnids incapable of swimming after 24 and 48 [h] of exposure in [%], n = 20, 4 replicates

IMMOBILIZATION [%]
Nom. Conc. [mg/L]	24 [h]					48 [h]
	1	2	3	4	MV	1	2	3	4	MV
1000	0	0	0	0	0	0	0	0	0	0
580	0	0	0	0	0	0	0	0	0	0
320	0	0	0	0	0	0	0	0	0	0
180	0	0	0	0	0	0	0	0	0	0
100	0	0	0	0	0	0	0	0	0	0
Contr.	0	0	0	0	0	0	0	0	0	0

MV = Mean value

Nom. Conc. = nominal concentration

Contr. = control group

 

Water parameters at the beginning of the test

(measured in one replicate per concentration and control)

Nom. Conc. [mg/L]	pH-value	O2-concentration [mg/L]
1000	8.06	8.2
580	8.06	8.1
320	8.04	8.2
180	7.99	8.2
100	7.91	8.3
Contr.	7.76	8.2

 

Water parameters at the end of test (48 [h]) (measured in all replicates)

Nom. Conc. [mg/L]	pH-values				Oxygen [mg/L]
	No. of replicate				No. of replicate
	1	2	3	4	1	2	3	4
1000	7.56	7.56	7.55	7.57	8.0	7.8	7.9	7.8
580	7.52	7.54	7.56	7.53	8.0	8.0	8.0	7.9
320	7.51	7.50	7.52	7.51	7.9	8.0	8.0	8.0
180	7.44	7.45	7.48	7.48	7.8	7.9	8.0	7.9
100	7.43	7.42	7.43	7.43	8.0	8.0	8.0	8.1
Contr.	7.26	7.29	7.32	7.31	8.0	8.1	8.0	8.1

 

Water parameter of the dilution water (Date: June 28, 1995)

pH-value	7.93
Oxygen	8.2 [mg/L]
Temperature	23.3 [°C]
Conductivity	674 [μS/cm]
Total hardness	15 [°dH]

 

Validity criteria fulfilled:

yes

Conclusions:

The EC0 was ≥ 1000 (mg/L) and the EC100 was > 1000 (mg/L) after 48 h of exposure.
The NOEC (48 h) was determined as ≥ 1000 (mg/L)a (nominal concentration).

Executive summary:

The acute immobilisation (EC50) of Lowinox WSP to Daphnia magna (STRAUS) was conducted according to OECD Guideline No. 202 I from June 28 to 30, 1995 at the DR.U.NDACK-LABORATORIUM FÜR ANGEWANDTE BIOLDGIE in 31137 Hildesheim, FRG.

 

Test substance was the product Lowinox WSP with 96.7 (%) Methylen-bis-(2-hydroxy-3-a-methyl cyclohexyl-4-methyl-benzol) as active ingredient.

Batch number of the tested sample was 5E049. No carrier for dissolving the test substance was used.

 

The test method was static. The test substance was applied once at test begin.

A static test was conducted with the nominal concentrations:

100 - 180 - 320 - 580 - 1000 (mg/L) selected on the basis of a preliminary static test.

Duration of the test was 48 hours.

20 test organisms were exposed to each test concentration and control.

 

No Effects and no mortality was seen in the nominal test concentrations.

The concentration with no effect for Lowinox WSP with respect to mobility of Daphnia magna was found to be ≥ 1000 (mg/L) after 48 (h) of exposure (nominal concentration).

The EC values are summarized in Table 1 (see below).

 

The concentrations of the active ingredient were determined in all concentrations at test start and test end. The mean recovery rate of the product at the test beginning was 63 (%) and at test end 89 (%).

Because the recovery rates of the active ingredient were between 73 and 109 % at the end of the test, all results are given as nominal concentrations.

 

Table 1. EC values (basing on nominal concentrations)

EC	[mg/L]
	Test duration [h]
	24	48
EC0	≥1000	≥1000
EC50	>1000	>1000
EC100	>1000	>1000

 

A reference test was carried out with potassium dichromate as reference substance and the reference toxicity determined. The EC50 value of the reference substance at 1.3 (mg/L) after 24 (h) was within the prescribed concentration range of 0.9 to 1.9 (mg/L).

Water quality parameters of pH and dissolved O2 measured at 0 and 48 (h) were determined to be within the acceptable limits.

The quality criteria of the test guideline were fullfiled.

Description of key information

Key value determined by laboratory testing to OECD guideline 202. 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1 000 mg/L

Additional information

The acute daphnia toxicity was determined for the test article Lowinox WSP.

Daphnia magna was used as test organism.

The acute toxicity test was performed with 5 concentration levels between 100 - 1000 (mg/L). All concentrations were analytically determined.

The EC0 was ≥ 1000 (mg/L) and the EC100 was > 1000 (mg/L) after 48 h of exposure.

During the test no mortality was observed at the tested concentrations.

The NOEC (48 h) was determined as ≥1000 (mg/L) (nominal concentration).