3-cyano-3,5,5-trimethylcyclohexanone

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:

1998-12-01 to 1998-12-10

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions: No analytical determination of test substance concentrations. However, the DOC of the stock and test solutions (after 72 hours) was analysed.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-cyano-3,5,5-trimethylcyclohexanone

EC Number:

411-490-4

EC Name:

3-cyano-3,5,5-trimethylcyclohexanone

Cas Number:

7027-11-4

Molecular formula:

C10H15NO

IUPAC Name:

3-cyano-3,5,5-trimethylcyclohexanone

Details on test material:

Test substance: Isophorone nitrile of Degussa AG, batch no. 25.9.98/ANT, purity: 99.8 %

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- at the start (0 hous) and the end of the study (72 hours)

Test solutions

Vehicle:

no

Details on test solutions:

STOCK AND TEST SOLUTION AND THEIR PREPARATION
- Dispersion: Crushing of test substance in mortar, weighing into  measuring flask, addition of water, ultrasonic treatment (12.5 g/l: 40  min; 1.25 g/l and 2.5 g/l: 20 min; 0.125 g/l: 5 min), stirring for 1-2  hours, filtration (sterile)
- Dilution water: Ultrapure water
- Concentration of stock solution: 12.5 g/l; 1.25 g/l; 0.125 g/l  (screening test). 2.5 g/l (main test)

STABILITY OF THE TEST CHEMICAL SOLUTIONS
- 12.5 g/l was above the  solubility as indicated by (1) turbidity upon addition of mineral salt solution and (2) TOC analysis

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISMS
- Strain: Scenedesmus subspicatus CHODAT (86.81 SAG)
- Source/supplier: Origin: Institute for Plant Sciences, University of  Göttingen (Germany); further bred inhouse
- Method of cultivation: SOP S 013 of the Institut Fresenius Group
- Pretreatment: Pre-culture with approx. 100,000 cells/ml in growth  medium 3 days in advance
- Initial cell concentration: approx. 60,000 cells/ml

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

- Test temperature: 22.6-23.8 °C (mean 23.23 °C)

pH:

- pH: Initially adjusted to 8 before adjustment of volume.   
- 8.10-8.24 (0 hours); 8.35-10.97 (72 hours)

Nominal and measured concentrations:

100; 1000; 10,000 mg/l (screening test, 5 replicates)   
125; 250; 500; 1000; 2000 mg/l (main test, 7 replicates)

Details on test conditions:

GROWTH/TEST MEDIUM CHEMISTRY   
Stock solutions in ultrapure water:   
(1) 1.5 g NH4Cl/L; 1.2 g MgCl2 x 6 H2O/L; 1.8 g CaCl2 x 2 H2O/L; 1.5 g MgSO4 x 7 H2O/L; 0.16 g KH2PO4/L 
(2) 0.08 g FeCl3 x 6 H2O/L; 0.10 g Na2EDTA x 2 H2O/L   
(3) 185 mg H3BO3/L; 415 mg MnCl2 x 4 H2O/L; 3 mg ZnCl2/L; 1.5 mg CoCl2 x 6 H2O/L; 0.01 mg CuCl2 x 2 H2O/L; 7 mg Na2MoO4 x 2 H2O/L  
(4) 1.0 g NaHCO3 (solid)   
880 ml ultrapure water + 100 ml (1) + 10 ml (2) + 10 ml (3) + (4),  equilibrated with air, sterilization by ultrafiltration (0.45 µm), further dilution 
1:10 for test solutions

TEST SYSTEM
- Exposure vessel type: 50 ml glass cylinders with 50 ml test solution and covered with a plexiglass lid
- Number of replicates: 5-7
- Intensity of irradiation: >= 120 µE/m2s (approx. 8000 lux)
- Photoperiod: Not reported, probably 24 hours/day
- Other relevant factors: Stirring for 15 min/hour

TEST PARAMETER
- Cell density as measured by extinction at 578 nm

CONTROLS
- algae control (no test substance, 7 replicates),  sterile control (no algae, 3 replicates per concentration), blank control (no test substance, no algae, 3 replicates)

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

78 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

105 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

350 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC100

Effect conc.:

1 600 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

110 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

156 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

580 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC100

Effect conc.:

3 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

According to the DOC measurements (via LiquiTOC, Foll-Heraeus) the test substance recovery in the stock and the test solutions was between 101% and 112%. The inhibition of the biomass production and the growth rate of the algae was calculated according to OECD 201 using MS Excel, version 5.0.

Results with reference substance (positive control):

No reference substance was tested

Any other information on results incl. tables

RESULTS
- Nominal/measured concentrations: 
  Nominal   0 h: TOC => measured   72 h: TOC => measured
  ------------------------------------------------------
   125 mg/l    84.8 =>  117 mg/l      95.03 =>  131 mg/l
   250 mg/l   178.9 =>  246 mg/l     182.40 =>  251 mg/l
   500 mg/l   343.5 =>  473 mg/l     368.68 =>  507 mg/l
  1000 mg/l   716.5 =>  986 mg/l     736.43 => 1013 mg/l
  2000 mg/l  1444.5 => 1987 mg/l    1465.63 => 2016 mg/l
  ------------------------------------------------------

- Cell density data: 
  Concentration  24 hours    48 hours    72 hours     EbC      ErC
  ------------------------------------------------------------------
  Control scr.    299,000     915,000   2,800,000     0.0 %    0.0 %
    100 mg/l      323,000   1,110,000   2,990,000   -12.6 %    0.8 %
   1000 mg/l      145,000     187,000     230,000    87.3 %   66.4 %
  10000 mg/l     --------------- not determined --------------------
  Control main    302,000     872,000   2,650,000     0.0 %    0.0 %
    125 mg/l      305,000     793,000   2,600,000     4.4 %    2.7 %
    250 mg/l      231,000     640,000   1.360,000    40.5 %   20.9 %
    500 mg/l      144,000     276,000     426,000    79.9 %   50.1 %
   1000 mg/l      131,000     177,000     201,000    89.5 %   70.3 %
   2000 mg/l       98,700     123,000     124,000    94.8 %   83.4 %
  ------------------------------------------------------------------
  "Control" refers to algae control.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The EbC50 (72 hours) was determined as 350 mg/L indicating that the test substance poses to significant hazard to algae

Executive summary:

The growth inhibition of isophorone nitrile on the alga Scenedesmus subspicatus was evaluated in a 72 -h growth inhibition test by Fresenius Institute (1999) according to OECD TG 201. Five concentrations ranging from 125 mg/L to 2000 mg/L (nominal) were tested. The following endpoints were derived:

EbC50 (72 hours): 350 mg/L

ErC50 (72 hours): 580 mg/L

NOEC (72 hours, based on cell numbers): 78 mg/L

The study was assessed as "reliable with restrictions".