Carbamic acid, N,N'-methylenebis(4,1-cyclohexanediyl)bis-, C,C'-dibutyl ester

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:

2004-01-20 to 2004-01-30

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

31 July 1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 07 June 1984

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-Methylenebis(N-butoxycarbonylcyclohexanamine)

EC Number:

619-422-7

Cas Number:

99305-42-7

Molecular formula:

C23 H42 N2 O4

IUPAC Name:

4,4'-Methylenebis(N-butoxycarbonylcyclohexanamine)

Details on test material:

- Test substance: 4,4'-Methylenebis(N-butoxycarbonylcyclohexanamine) of Degussa AG
- Batch 4/032, purity approximately 95 % determined 04-Nov-2003
- White solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

MONITORING OF TEST SUBSTANCE CONCENTRATION: Analysis of samples taken at  study start and after 24 and 72 hours from all concentrations

Test solutions

Vehicle:

no

Details on test solutions:

STOCK AND TEST SOLUTION AND THEIR PREPARATION
- Dilution water:algal culture medium
- Concentration in stock solution: 100 mg/l nominal
- Other procedures: Stirring for approx. 24 hours followed by filtration  (0.45 µm pore size)

STABILITY OF THE TEST CHEMICAL SOLUTIONS
- Visually clear solutions

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISMS
- Strain: Pseudokirchneriella subcapitata SAG Strain No. 61.81, former  name Selenastrum capricornutum
- Source/supplier: Origin Albrecht-von-Haller-Institute for Plant  Sciences, University of Göttingen (Germany)
- Method of cultivation: Inhouse culture according to appendix 1 of  relevant test guideline
- Controls: Concurrent six replicates without test substance
- Initial cell concentration: approx. 10,000 cells/ml

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

23-23.5 °C

pH:

pH 7.7-7.8 (start), 9.1-10.0 (end); not adjusted

Nominal and measured concentrations:

- Concentrations: 6.25; 12.5; 25; 50; 100 mg/l (nominal)

Details on test conditions:

TEST SYSTEM
- Exposure vessel type: Culture flasks (unspecified) with 100 ml test  solution per replicate
- Number of replicates: 3
- Intensity of irradiation: 6,000-10,000 Lux, 400-700 nm
- Photoperiod: Not reported, probably 24 hours/day
- Other relevant factors: Continuous stirring

TEST PARAMETER: Cell density (Casy Coulter Counter)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate, at least  twice a year

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

106.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 31.61-51.11 mg/l

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

680 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 324.54-7514.22 mg/l

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

42.87

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 31.61-51.11 mg/L

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

123.71 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 108.52-152.77 mg/L

Details on results:

No visible changes of the shape of the algae were noticed either in the control or in the treatment groups (microscope).

The percentage of the nominal concentrations determined analytically (i.e. the ratio measured/nominal) decreased with increasing nominal 
concentration.  A correlation between the dose-dependent toxicity recorded and the low  analytical concentrations could not be established. 
Therefore evaluation was based on nominal concentrations.

Results with reference substance (positive control):

- no data

Any other information on results incl. tables

RESULTS: 
- Nominal/measured concentrations (mean value): 
    6.25 mg/l: 0.01410 mg/l = 0.23 %
   12.5 mg/l:  0.00954 mg/l = 0.08 %
   25 mg/l:    0.01263 mg/l = 0.05 %
   50 mg/l:    0.01607 mg/l = 0.03 %
  100 mg/l:    0.01996 mg/l = 0.02 %
  
- Cell density data: 
  Concentration  24 hours   48 hours    72 hours
  ----------------------------------------------
  Control          50,257    319,233   1,024,596
    6.25 mg/l      50,653    317,922   1,016,211
   12.5 mg/l       50,888    325,556     955,978
   25 mg/l         51,847    324,578     972,589
   50 mg/l         48,998    273,689     885,267
  100 mg/l         42,464    165,967     662,633
  ----------------------------------------------
 

- Percentage reduction of biomass (area under curve):
  Concentration  24 hours   48 hours    72 hours
  ----------------------------------------------
    6.25 mg/l       -1          0           1
   12.5 mg/l        -2         -2           3
   25 mg/l          -4         -2           2
   50 mg/l           3         12          14
  100 mg/l          19         43          40
  ----------------------------------------------

- Growth curves: Percentage reduction of growth rate:
  Concentration  24 hours   48 hours    72 hours
  ----------------------------------------------
    6.25 mg/l        0          0           0
   12.5 mg/l        -1         -1           1
   25 mg/l          -2          0           1
   50 mg/l           2          4           3
  100 mg/l          10         19           9

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The lowest 72h EC50 value was based on cell numbers and was determined as 123.71 mg/L indicating that the test substance does not pose a
significant hazard to aquatic algae. However, the results have to be treated with caution because of the very low solubility of the test substance which
ranges from 0.01 to 0.02 mg/L.

Executive summary:

The growht inhibition effects of H12MDU on the alga Pseudokirchneriella subcapitata were investigated in a static test over a period of 72 hours according to EU method C.2 and OECD TGD 202. Five concentrations ranging from 6.25 mg/L to 100 mg/L were tested. The following endpoints were derived:

EC50 (cell numbers, 72 hours): 123.71 mg/L; CL: 108.52 -152.77 mg/L

EC50 (growth rate, 72 hours): 680 mg/L; CL: 324.54 -7514.22 mg/L

However, it has to be noted that due to the very low water solubility of the test substance the masured concentrations were only in a range of 0.23 to 0.02% of the nominal concentrations. A correlation between the dose-dependent toxicity recorded and the low  analytical concentrations could not be established. 

Therefore evaluation was based on nominal concentrations. For this reason, the results of the study have to be treated with caution.

The study was assessed as "reliable with restrictions".