3,5,5-trimethylhexanoic acid

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:

2010-04-09 to 2010-04-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Directive 92/69/EEC, C.3: Algal Inhibition Test, 1992

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EC) No 761/2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all concentrations sampled (The concentrations of the test item 3,5,5 Trimethylhexanoic acid were determined in the duplicate test medium samples from the nominal test concentrations of 10 to 100 mg/L. The samples from the nominal test concentrations of 1.0 and 3.2 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test. From the control samples, one of the duplicate samples was analyzed from the corresponding sampling times. )
- Sampling method: duplicate samples
- Sample storage conditions before analysis: All samples were stored protected from light and deep frozen (approx. -20 °C)

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: stock solution = highest nominal concentration of 100 mg/L (dissolving 33.16 mg of the test item completely in 332 mL of test water stirring intensely for 15 minutes at room temperature); the test media of the lower test concentrations prepared by diluting stock solution; preparation just before the start of the test.
- Controls: negative
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: Strain No. 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany); cultivated at Harlan Laboratories under standardized conditions
- Age of inoculum (at test initiation): 4 d, exponential growth phase
- Method of cultivation:

ACCLIMATION
- Acclimation period: not applicable

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol (= 24 mg/L as CaCO3)

Test temperature:

22 °C

pH:

test start: 7.1 to 8.2
test end: 6.9 to 8.9

Nominal and measured concentrations:

nominal: 0 (control), 1.0, 3.2, 10, 32 and 100 mg/L
measured: recovery rate: 91 to 103%

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed (covered with glass dish)
- Material, size, headspace, fill volume: 50 mL Erlenmeyer flasks, fill volume: 15 mL
- Aeration: no
- Initial cells density: 10000 cells/mL
- Control end cells density:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water according to test guidelines

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: approximately 7700 Lux (range: 7220 to 8290 Lux); fluorescent tubes (Philips TLD 36W-1/840)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter
- Chlorophyll measurement: no
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: no data
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

81 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 76 - 86 mg/L

Remarks:

test item concentrations analytically confirmed

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

45 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 35 - 53 mg/L

Remarks:

test item concentrations analytically confirmed

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

51 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: yield (biomass)

Remarks on result:

other: 95% CL: 48 to 55 mg/L

Remarks:

test item concentrations analytically confirmed

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: growth rate and yield

Remarks on result:

other: test item concentrations analytically confirmed

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: shape and size of the algal cells were obviously not affected (visual inspection of cells in 32 m/L treatment)
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not applicable
- Effect concentrations exceeding solubility of substance in test medium: no

Biomass of Algae

Nominal test item concentration (mg/L)		Biomass of algae*
		24 hours	48 hours	72 hours
Control	Mean	10.6	64.6	299.9
	SD	1.0	4.5	20.3
1.0	Mean	10.6	66.6	313.5
	SD	1.0	6.1	31.3
3.2	Mean	9.5	59.6	300.3
	SD	0.8	2.5	15.5
10	Mean	9.7	59.3	303.1
	SD	0.7	5.0	23.8
32	Mean	9.6	56.0	269.8
	SD	0.7	0.9	3.7
100	Mean	3.7	8.6	11.6
	SD	0.6	1.4	3.6

 

SD:     Standard deviation

*:        The biomass was determined by fluorescence measurement (at least duplicate measurements per replicate) and is given as relative fluorescence units (x 103). At the start of the test, the initial cell density was 10000 algal cells/mL, corresponding to 2.30 x 103 relative fluorescence units.

 

 

Average Growth Rates (µ)

Nominal test item concentration	Average growth rate µ (day-1) and inhibition of µ (Ir)
	0-24 h		0-48 h		0-72 h
(mg/L)	µ	Ir (%)	µ	Ir (%)	µ	Ir (%)
Control	1.53	0.0	1.67	0.0	1.62	0.0
1.0	1.53	0.2	1.68	-0.9	1.64	-0.9
3.2	1.42	7.3	1.63	2.4	1.62	0.0
10	1.44	5.7	1.62	2.6	1.63	-0.2
32	1.43	6.4	1.60**	4.3	1.59**	2.1
100	0.45*	70.3	0.65**	60.9	0.53**	67.4

 

*:        mean value significantly lower than in the control
(according to Williams t-test, one-sided smaller,a= 0.05)

**:       mean value significantly lower than in the control
(according to Welch t-test, one-sided smaller,a= 0.05)

 

 

Yield(Y)

Nominal test item concentration	Yield Y (x 103) and inhibition of Y (Iy)
	0-24 h		0-48 h		0-72 h
(mg/L)	Y	Iy(%)	Y	Iy(%)	Y	Iy(%)
Control	8.3	0.0	62.3	0.0	297.6	0.0
1.0	8.3	0.4	64.3	-3.2	311.2	-4.6
3.2	7.2	13.6	57.3	8.1	298.0	-0.1
10	7.4	10.8	57.0*	8.6	300.8	-1.1
32	7.3	12.1	53.7*	13.9	267.5*	10.1
100	1.4*	83.8	6.3*	90.0	9.3*	96.9

 

*:        mean value significantly lower than in the control
(according to Williams t-test, one-sided smaller,a= 0.05)

 

 

Section-by-Section Growth Rates

Nominal test item concentration	Section-by-section growth rates (day-1) and inhibition of the growth rates (Ir)
	0-24 h		24-48 h		48-72 h
(mg/L)	µ	Ir(%)	µ	Ir(%)	µ	Ir(%)
Control	1.53	0.0	1.81	0.0	1.53	0.0
1.0	1.53	0.2	1.84	-1.8	1.55	-0.9
3.2	1.42	7.3	1.84	-1.7	1.62	-5.3
10	1.44	5.7	1.81	0.0	1.63	-6.4
32	1.43	6.4	1.76	2.4	1.57	-2.5
100	0.45	70.3	0.85	52.9	0.28	81.6

 

 

pH Values in theTreatments

Nominal test item concentration	pH values
(mg/L)	Start	End
Control	8.2	8.8
1.0	8.0	8.9
3.2	8.0	8.7
10	7.9	8.7
32	7.8	8.5
100	7.1	6.9

 

The biological results of the study were as follows: 

Parameter	Growth rate	Yield
(0-72 h)
EC10  (mg/L)	45	32
95% confidence interval	35 - 53	30 – 34
EC20  (mg/L)	55	38
95% confidence interval	46 - 62	35 – 40
EC50  (mg/L)	81	51
95% confidence interval	76 - 86	48 – 55
NOEC (mg/L)	10	10
LOEC (mg/L)	32	32

Validity criteria fulfilled:

yes

Conclusions:

The 72-h ErC50 was determined to be 81 mg/L, the 72-h ErC10 was 45 mg/L and the 72-h NOEC was 10 mg/L (OXEA, 2010).

Executive summary:

The growth inhibiting effect of 3,5,5 -trimethylhexanoic acid was investigated on the green alga Pseudokirchneriella subcapitata in a 72 -h study according to OECD TG 201. The following nominal concentrations were set up: 0, 1.0, 3.2, 10, 32 and 100 mg/L. The test substance concentrations were analytically verified on days 0 and 3. The recovery rates were between 91 and 103%, therefore the results are based on nominal concentrations. The physico-chemical parameters pH, temperature and light intensity were within the recommended ranges. The validity criteria of OECD TG 201 (March 2006) were fulfilled. Based on nominal concentrations (analytically confirmed), the 72 -h EbC50 was 51 mg/L and the ErC50 was 81 mg/L. The 72-h ErC10 was 45 mg/L and the NOEC was 10 mg/L for growth rate and yield. Based on the EC50 3,5,5 -trimethylhexanoic acid is acutely harmful to algae.

Description of key information

Pseudokirchneriella subcapitata: 72-h ErC50: 81 mg/L (OECD 201; OXEA, 2010)

Key value for chemical safety assessment

EC50 for freshwater algae:

81 mg/L

EC10 or NOEC for freshwater algae:

45 mg/L

Additional information

The growth inhibiting effect of 3,5,5 -trimethylhexanoic acid was investigated on the green alga Pseudokirchneriella subcapitata in a 72 -h study according to OECD TG 201. The following nominal concentrations were set up: 0, 1.0, 3.2, 10, 32 and 100 mg/L. The test substance concentrations were analytically verified on days 0 and 3. The recovery rates were between 91 and 103%, therefore the results are based on nominal concentrations. The physico-chemical parameters pH, temperature and light intensity were within the recommended ranges. The validity criteria of OECD TG 201 (March 2006) were fulfilled. Based on nominal concentrations (analytically confirmed), the 72 -h EbC50 was 51 mg/L and the ErC50 was 81 mg/L. The 72-h ErC10 was 45 mg/L and the NOEC was 10 mg/L for growth rate and yield. Based on the EC50, 3,5,5 -trimethylhexanoic acid is acutely harmful to algae.