4,4'-methylenebis(cyclohexylamine)

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989-04-19 - 1989-04-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: guideline study

Qualifier:

according to guideline

Guideline:

other: Directive 79/831/EEC, Annex V, Part C (1984)

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

- Method: Stock solution has been prepared
- Concentration of the stock solution: 100 mg/L nominal. The stock solution was stirred for 1020 min at 293 K

Test organisms (species):

Daphnia magna

Details on test organisms:

- Source: Daphnia magna Straus was originally supplied from "Institut National de Recherche Chimique appliquee, France", and has been breeded from 1978 in the Laboratories of BASF.
- age of animals: 2-24 h

Breeding conditions:
Water
Hardenss: 2.76 +- 0.5 mmol/l
Ks to pH 4.3 : 6.96 +- 6.1 mmol/l
Mol realtion Ca : Mg; approx. 4: 1
Oxygen content: > 2 mg/1
Temperature range: 292.6 - 294.6 K
Feeding once daily green algae
Day : Night rhythm 16:8 hours
Light intensity: approx.. 5 Mikroeinstein / (m*m*s); 466- 756 nm

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

2.75 mmol/L

Test temperature:

- temperature: 20-22 °C (292-294 K)

pH:

-range of pH at start: 8.07 (6.25, 12.5 mg/L) - 9.05 (100 mg/L)
-range of pH after 48 h: 7.95 (100 mg/L, control) - 8.06 (1.56 mg/L)

Dissolved oxygen:

-range of O2 (mg/L) at start: 8.62 (100 mg/L) - 9.33 (0.781 mg/L)
-range of O2 (mg/L) after 48 h: 8.7 (100 mg/L) - 8.93 (1.56 mg/L)

Conductivity:

663µS/cm

Nominal and measured concentrations:

- test concentrations (nominal): 0.781, 1.56, 3.12, 6.25, 12.5, 25.0, 50.0, 100 mg/L

Details on test conditions:

- dilution water: source: tap water; pretreatment steps: (1) 6 µm- and charcoal-filtration; (2) H2SO4 was added to reduce alkalinity up to pH 4.3; (3) distilled water was added to reduce water-hardness; (4) water was aerated (oil-free air) unil saturated with oxygen; (5) water was stored for at least 24 h for stabilization. Specifications measured at test start: water-hardness: 2.75 mmol/L, alkalinity up to pH 4.3: 0.94 mmol/L, pH: 8.0, conductivity: 663 µSiemens/cm

- O2-content: > 2 mg/L
- Photoperiod: 16:8 hours
- illumination: diffuse light
- test volume: 10 ml
- test vessels: test tubes (glass) with flat bottom (nominal volume 20 mL)
- replicates: 4 per concentration
- volume/animal: 2 mL
- number of animals/vessel: 5
- total number of animals/conc.: 20
- observation times: visually after 0, 3, 6, 24 and 48 h
- observation parameters: swimming ability, pH, oxygen
- test was performed in non-sealed vessels

Procedures to determine EC-values after 48 h:
- EC50: moving average (Sachs L., 1974; Thompson W.R., 1947)
- EC0: highest concentration tested at which <= 10 % of the animals were immobile
- EC100: lowest tested concentration at which 100 % of the animals were immobile

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

3.12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

9.24 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 6.55 - 11.93 mg/L

Duration:

24 h

Dose descriptor:

EC0

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

19.65 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 15.65 - 23.65 mg/L

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Number of mobile test animals after exposure (48 h) to
various test concentrations:
concentration (mg/L)  mobile Daphnids
      0.781                 20
      1.56                  20
      3.12                  20
      6.25                  17
     12.5                    4
     25.0                    0
     50.0                    0
    100.0                    0
   control                  20

Conclusions:

Based on the nominal initial concentrations of the test item, the 48-hour EC50 for Daphnia magna for immobilisation was 9.24 mg/L (95% CL: 6.55 – 11.93 mg/L).

Executive summary:

The toxicity of the test item was determined according Directive 79/831/EEC, Annex V, Part C (1984).

The study was conducted under static conditions over a period of 48 hours with eight nominal test concentrations: test concentrations (nominal): 0.781, 1.56, 3.12, 6.25, 12.5, 25.0, 50.0, 100 mg/L prepared from a 100 mg/L stock solution.

 

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control in a 20 ml glass beaker containing 10 mL of test solution. Oxygen saturation and pH values were monitored at the beginning and termination of the study

Based on the nominal initial concentrations of the test item, the 48-hour EC50 for Daphnia magna for immobilisation was 9.24 mg/L (95% CL: 6.55 – 11.93 mg/L).

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-07-11 to 2002-08-19

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Test item concentration and test item stability not verified.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

1984

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Test concentrations:
6 nominal concentrations of 0.625; 1.25; 2.5; 5; 10 and 20 mg/L prepared from a 102 mg/L compound stock solution prepared in standard MHRW water. Prior to initiation the stock solution was pH adjusted to approximately neutral with 1.0 N hydrochloric acid.

Test organisms (species):

Daphnia magna

Details on test organisms:

- Common name: Daphnia magna
- Age at study initiation (mean and range, SD): Less than 24 hours old.
- Stage and instar at study initiation: Neonates
- Method of breeding: In-house culture in standard reconstituted MHRW water
- Source: In-house culture from Biological Monitoring, Inc. (BMI)
- Feeding during test: No
ACCLIMATION
- Acclimation period: Not needed as culturing and testing water have the same composition.

Test type:

static

Water media type:

other: standard reconstitued MHRW water.

Limit test:

no

Total exposure duration:

48 h

Hardness:

Control: 92 mg/L CaCO3

Test temperature:

20 (+/-) 1 deg C

pH:

pH of stock solution was adjusted from 9.77 to 7.19 using 1.0 N HCl.

pH 0 mg/ L: 0h - 8,0; 48 h - 8,1
0,625 mg/ L: 0h -7,9; 48 h - 7,9
1,25 mg/ L: 0h - 7,8; 48 h - 7,8
2,5 mg/ L: 0 h - 7,8; 48 h - 7,8
5 mg/ L: 0 h - 7,8; 48 h - 7,8
10 mg/ L: 0 h - 7,7; 48 h - 7,8
20 mg/ L: 0 h - 7,8; 48 h - 7,9

Dissolved oxygen:

Dissolved Oxygen (mg/ L)
0 mg/ L: 0h - 7,8; 48 h - 7,7
0,625 mg/ L: 0h -7,7; 48 h - 7,7
1,25 mg/ L: 0h - 7,8; 48 h - 7,7
2,5 mg/ L: 0 h - 7,8; 48 h - 7,7
5 mg/ L: 0 h - 7,8; 48 h - 7,6
10 mg/ L: 0 h - 7,8; 48 h - 7,7
20 mg/ L: 0 h - 7,7; 48 h - 7,6

Salinity:

control: 64 mg/L

Conductivity:

Conductivity (µmHOS)
0 mg/ L: 0h - 290; 48 h - 290
0,625 mg/ L: 0h -290; 48 h - 290
1,25 mg/ L: 0h - 290; 48 h - 290
2,5 mg/ L: 0 h - 280; 48 h - 290
5 mg/ L: 0 h - 280; 48 h - 290
10 mg/ L: 0 h - 290; 48 h - 300
20 mg/ L: 0 h - 300; 48 h - 310

Nominal and measured concentrations:

0.625, 1.25, 2.5, 5, 10, 20 mg/L nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 ml Polypropylene beakers
- Type (delete if not applicable): non-sealed vessels
- Material, size, headspace, fill volume: yes
- Volume of solution: 35 ml
- Aeration: no
- - Renewal rate of test solution (frequency/flow rate): no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstitued MHRW water.
- Hardness: Control: 92 mg/L CaCO3
- Alkalinity: pH= 8 in control
- Conductivity: 290 µmHOS
- Salinity: 64 mg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: 0 hour and 48 hours
OTHER TEST CONDITIONS
- Adjustment of pH: yes. A 102 mg/L stock solution of PACM in water was prepared and the pH was adjusted from 9.77 to 7.19 using 1.0 N HCl. The pH adjusted stock solution was used to prepare the test concentrations.
- Photoperiod:16h light/8h dark
- Light intensity: not specified
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
VEHICLE CONTROL PERFORMED: no
RANGE-FINDING STUDY: Yes
- Test concentrations: 0.0098; 0.098; 0.98; 9.8; 98 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Sodium chloride

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

7.07 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 6.05-8.27 95% CI

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: Erratically swimming behaviour of daphnia in the 10 mg/L group at the end of the test
- Observations on body length and weight: no
- Other biological observations:
- Mortality of control: no
- Other adverse effects control: no
- Immobilisation of control: no
- Abnormal responses: At the end of test, the surviving daphnids in the 10 mg/L group were swimming erratically.
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

The 48-hour EC50 of sodium chloride for Daphnia magna was 5.278 g/L with 95% confidence limit of 4.809 and 5.792 g/L. This value fell within the acceptable limits established with the laboratory's reference toxicant chart, indicating that the D. mgna used in this study were suitable for testing.

Reported statistics and error estimates:

The endpoint for the study was EC50, the effect concentration to fifty percent of the test
organisms. The EC50 values were generated fkom software that using Binomial, Moving
Average, Probit, and/or Spearman Karber statistical methods.

Table 1: Number of Mobile Organisms at 48 Hours

Concentration (mg/L)	Mobile Daphnids
0.625	20
1.25	20
2.5	20
5	18
10	2
20	0

Validity criteria fulfilled:

yes

Remarks:

Negative controls demonstrated < 0% immobilization and dissolved O2 concentration at the conclusion was 7.9 mg/l or higher.

Conclusions:

Based on the nominal concentrations of the test item with pH adjusted to 7.2., the 48-hour EC50 for Daphnia magna for immobilisation was 7.07 mg/L (95% CL: 6.046 - 8.270).

Executive summary:

The toxicity of the test item was determined according to OECD 202 guideline and the study was performed following OECD Good Laboratory Practices (GLP).

The study was conducted under static conditions over a period of 48 hours with six nominal test concentrations: 0.625, 1.25, 2.5, 5, 10, 20 mg/L prepared from a 102 mg/L compound stock solution prepared in standard MHRW water. Prior to initiation the stock solution was pH adjusted to approximately neutral with 1.0 N hydrochloric acid. These chosen concentrations were based on the results from a range finding toxicity with the test item.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control in a 50 ml polypropylene beaker containing 35 mL of test solution. The neonates were not fed for the test duration and the test temperature was 20 ±1°C. Water parameters were monitored during the whole study.

Based on the nominal initial concentrations of the test item, the 48-hour EC50 for Daphnia magna for immobilisation was 7.07 mg/L (95% CL: 6.046 - 8.270).

The validity criteria of the test guideline were fulfilled.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-07-11 to 2002-08-19

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Test item concentration and test item stability were not verified.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

1984

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Test concentrations:
6 nominal concentrations of 0.625; 1.25; 2.5; 5; 10 and 20 mg/L prepared from a 102 mg/L compound stock solution prepared in standard MHRW water.
pH of stock solution was not adjusted, and was measured at pH 7.9 - 9.7.

Test organisms (species):

Daphnia magna

Details on test organisms:

- Common name: Daphnia magna
- Age at study initiation (mean and range, SD): Less than 24 hours old.
- Stage and instar at study initiation: Neonates
- Method of breeding: In-house culture in standard reconstituted MHRW water
- Source: In-house culture from Biological Monitoring, Inc. (BMI)
- Feeding during test: No
ACCLIMATION
- Acclimation period: Not needed as culturing and testing water have the same composition.

Test type:

static

Water media type:

other: standard reconstitued MHRW water.

Limit test:

no

Total exposure duration:

48 h

Hardness:

Control 92 mg/L CaCO3

Test temperature:

20 (+/-) 1 deg C

pH:

pH 0 mg/ L: 0h - 8,0; 48 h - 8,0
0,625 mg/ L: 0h -7,6; 48 h - 7,7
1,25 mg/ L: 0h - 7,7; 48 h - 7,8
2,5 mg/ L: 0 h - 7,9; 48 h - 7,8
5 mg/ L: 0 h - 8.0; 48 h - 7.7
10 mg/ L: 0 h - 8,4; 48 h - 7,8
20 mg/ L: 0 h - 8,8; 48 h - 7,9

Dissolved oxygen:

Dissolved Oxygen (mg/ L)
0 mg/ L: 0h - 7,8; 48 h - 7,8
0,625 mg/ L: 0h -7,8; 48 h - 7,6
1,25 mg/ L: 0h - 7,8; 48 h - 7,7
2,5 mg/ L: 0 h - 7,8; 48 h - 7,7
5 mg/ L: 0 h - 7,8; 48 h - 7,6
10 mg/ L: 0 h - 7,8; 48 h - 7,6
20 mg/ L: 0 h - 7,8; 48 h - 7,7

Salinity:

control: 60 mg/L

Conductivity:

Conductivity (µmHOS)
0 mg/ L: 0h - 300; 48 h - 290
0,625 mg/ L: 0h -280; 48 h - 280
1,25 mg/ L: 0h - 275; 48 h - 280
2,5 mg/ L: 0 h - 280; 48 h - 280
5 mg/ L: 0 h - 280; 48 h - 280
10 mg/ L: 0 h - 280; 48 h - 280
20 mg/ L: 0 h - 285; 48 h - 290

Nominal and measured concentrations:

0.625, 1.25, 2.5, 5, 10, 20 mg/L nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 ml Polypropylene beakers
- Type (delete if not applicable): non-sealed vessels
- Material, size, headspace, fill volume: yes
- Volume of solution: 35 ml
- Aeration: no
- - Renewal rate of test solution (frequency/flow rate): no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstitued MHRW water.
- Hardness: Control: 92 mg/L CaCO3
- Alkalinity: pH= 8 in control
- Conductivity: 300 µmHOS (0 hour)
- Salinity: 64 mg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: 0 hour and 48 hours
OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod:16h light/8h dark
- Light intensity: not specified
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
VEHICLE CONTROL PERFORMED: no
RANGE-FINDING STUDY: Yes
- Test concentrations: 0.0098; 0.098; 0.98; 9.8; 98 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

sodium chloride

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

6.84 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 5.669-8.252 95% CI

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: Erratically swimming behaviour of daphnia in the 5 mg/L group at the end of the test; all daphnia dead in 20 mg/L
- Observations on body length and weight: no
- Other biological observations:
- Mortality of control: no
- Other adverse effects control: no
- Immobilisation of control: no
- Abnormal responses: Erratically swimming behaviour of daphnia in the 5 mg/L group at the end of the test; all daphnia dead in 20 mg/L
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

The 48-hour EC50 of sodium chloride for Daphnia magna was 5.278 g/L with 95% confidence limit of 4.809 and 5.792 g/L. This value fell within the acceptable limits established with the laboratory's reference toxicant chart, indicating that the D. mgna used in this study were suitable for testing.

Reported statistics and error estimates:

The endpoint for the study was EC50, the effect concentration to fifty percent of the test
organisms. The EC50 values were generated from software using Binomial, Moving Average,
Probit, and/or Spearman Karber statistical methods.

Table 1: Number of Mobile Organisms at 48 Hours

Concentration (mg/L)	Mobile Daphnids
0.625	20
1.25	20
2.5	20
5	15
10	4
20	0

Validity criteria fulfilled:

yes

Remarks:

Negative controls demonstrated < 0% immobilization and dissolved O2 concentration at the conclusion was 7.9 mg/l or higher.

Conclusions:

Based on the nominal concentrations of the test item, the EC50 for immobilisation was 6.84 mg/L (95% CL: 5.669 - 8.252).

Executive summary:

The toxicity of the test item was determined according to OECD 202 guideline and the study was performed following OECD Good Laboratory Practices (GLP).

The study was conducted under static conditions over a period of 48 hours with six nominal test concentrations: 0.625, 1.25, 2.5, 5, 10, 20 mg/L prepared from a 102 mg/L compound stock solution prepared in standard MHRW water. These chosen concentrations were based on the results from a range finding toxicity with the test item.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration level and the control in a 50 ml polypropylene beaker containing 35 mL of test solution. The neonates were not fed for the test duration and the test temperature was 20 ±1°C. Water parameters were monitored during the whole study.

Based on the nominal initial concentrations of the test item, the 48-hour EC50 for Daphnia magna for immobilisation was was 6.84 mg/L (95% CL: 5.669 - 8.252).

The validity criteria of the test guideline were fulfilled.

Description of key information

The short-term EC50 value for aquatic invertebrates was based upon the 48 -hour EC50 7.64 mg/L, the geometric mean of three short-term toxicity assay EC50 values in Daphnia magna (6.84mg/L, 7.07 mg/L and 9.24 mg/L); each value was a 48-hour EC50 from a key study conducted in accordance with an approved guideline.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

7.64 mg/L

Additional information

The use of the geometric mean value to represent the three closely-clustered EC50 values was appropriate, in accordance with Section R.10.3.1.1 of Chapter R.10, Characterization of dose[concentration]-response for environment, of Guidance on information requirements and chemical safety assessment (ECHA, May 2008).

 

The 48-hour geometric mean EC50 of 7.64 mg/L for Daphnia magna was the representative value for the short-term toxicity of 4,4'-methylenebis(cyclohexylamine) to aquatic invertebrates. This value represented one of the three short-term aquatic toxicity values for each of three trophic levels (fish, aquatic invertebrates and aquatic algae and plants). This geometric mean EC50 was the lowest of the three short-term aquatic toxicity values from each of the three trophic levels and was used as the point of departure for the derivation of the PNECaquatic (freshwater).