Trinonyl benzene-1,2,4-tricarboxylate

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:

2000-12-12 to 2001-12-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

(adopted 1984)

Deviations:

no

Remarks:

(with regard to the updated guideline in 2006, the new parameters on coefficients of variations were not determined at the time the study had been conducted)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test concentrations, blank control, dispersant control
- Sampling method: 0.2 - 1.0 mL sample was taken from three replicates of each treatment group at test start (before seeding with algae) and after 72 hours of exposure (these samples were centrifuged before analysis)
- Sample storage conditions before analysis: No data

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: test substance stock solution prepared at 100 mg/L with HCO-50 as solubilizing agent; each of the tested solutions consisted of filtered sterilized stock solution and culture medium; each treatment groups comprised 4 test vessels (one used only for pH measurements)

- Eluate: OECD culture medium
- Controls: blank control (OECD culture medium), dispersant control (HCO-50)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): solubilizer, hydrogenated castor oil HCO-50 (100 mg/L)
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 100 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): all test solutions were of a pale milky white colour with no sedimentation

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: unicellular green algae
- Strain: Selenastrum capricornutum (ATCC-22662)
- Source (laboratory, culture collection): American Type Culture Collection
- Method of cultivation: shaking culture (100 rpm, AGP-150RL apparatus )

ACCLIMATION
- Acclimation period: three days prior test start
- Culturing media and conditions (same as test or not): OECD culture medium, yes (same as test conditions)
- Any deformed or abnormal cells observed: no deformed or abnormal cells

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.0 – 24.5 deg C

pH:

start of the test: 7.3 – 7.5
end of the test: 8.1 – 8.3

Dissolved oxygen:

no data

Nominal and measured concentrations:

Nominal concentrations: 0.79 mg/L, 1.57 mg/L, 3.13 mg/L, 6.25 mg/L, 12.5 mg/L, 25 mg/L and 50 mg/L
Measured concentrations:
- start of the test (0h): 0.72, 1.48, - (failed), 6.02, 13.6, 29.4 and 56.8 mg/L
- end of the test (72h): 0.67, 1.34, 2.85, 5.67, 14.8, 25.2 and 44.6 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flask (air permeable silicone stopper)
- Material, size, headspace, fill volume: 300 ml capacity
- Test solution volume: 100 ml (OECD culture medium)
- Aeration:
- Renewal rate of test solution (frequency/flow rate): N/A, static
- Initial cells density: 1 × 10^4 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes, OECD culture medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH: at the start and end of the test in one replicate of each treatment group; temperature/light intensity: once per day

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous lighting
- Light intensity and quality: 4000 – 5000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentration: Cell density measurements were performed by collecting 0.2 – 1.0 ml of test solution from each test vessel, which was mixed with electrolytic solution (Isoton II) to make a total volume of 20 ml, and counted by Coulter counter (particle counter); time intervals: at 24 hours, 48 hours and 72 hours
Effect parameters measured:
- Growth inhibition (EbC50) by comparison with the area under the growth curve
- Growth inhibition concentration by comparison with growth rate (ErC50)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Details on results: During 72 hours, the cell density increased by a factor of 132 for the blank and a factor by 129 for the dispersant control. For the lowest test concentration, the cell density increased by a factor of 128 and was similar compared to the controls. The cell density increased by a factor of 108 for the highest test concentration indicating a slight inhibition of growth. For the other five concentrations (1.57 mg/L, 3.13 mg/L, 6.25 mg/L, 12.5 mg/L, 25.0 mg/L), the cell density increased by a factor of 111 – 122.

- Observation of abnormalities (for algal test): no data
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: All test solutions were of a pale milky white colour with no sedimentation or other concerns.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EbC50: 0.41 mg/L (72h)

Reported statistics and error estimates:

Bartlett distribution test, ANOVA and Dunnett tests were performed using Yukms StatLight #3 (multi-group comparison).

Results growth inhibition of Selenastrum capricornutum exposed to the test substance

Nominal	Inhibition (%)
Concentration (mg/L)	Yield (Biomass)	Growth rate
Control	-	-
Dispersant control	4.29	-2.08
0.79	3.39	-0.20
1.57	7.37	-0.96
3.13	14.91	-0.13
6.25	17.44	2.83
12.5	14.91	5.36
25.0	17.22	3.19
50.0	7.19	21.48

Validity criteria fulfilled:

yes

Remarks:

(adopted 1984; 2006)

Conclusions:

The 72h-EbC50 (green algae, biomass) was > 50.0 mg/L and the 72h-NOECb was >= 50.0 mg/L; the 72hErC50 (green algae, growth rate) was > 50.0 mg/L and the 72h-NOECr was 25.0 mg/L

Executive summary:

Toxicity to green algae (Selenastrum capricornutum) was investigated during 72 hours of exposure according to OECD 201 (adopted 1984) and in compliance with GLP. As the test substance is of low water solubility, a dispersion agent was used to increase the test concentration in the test medium (hydrogenated castor oil HCO-50, 100 mg/L). The test was performed using seven test concentrations (nominal 0.79 mg/L, 1.57 mg/L, 3.13 mg/L, 6.25 mg/L, 12.5 mg/L, 25.0 mg/L and 50 mg/L). A blank and vehicle control were run in parallel.

During 72 hours, the cell density increased by a factor of 132 for the blank and a factor by 129 for the dispersant control. For the lowest test concentration, the cell density increased by a factor of 128 and was similar compared to the controls. The cell density increased by a factor of 108 for the highest test concentration indicating a slight inhibition of growth. For the other five concentrations (1.57 mg/L, 3.13 mg/L, 6.25 mg/L, 12.5 mg/L, 25.0 mg/L), the cell density increased by a factor of 111 – 122.

Actual measurements of the test concentration were within the range of ± 20% of the nominal concentration, and therefore effect concentrations were based on the nominal test concentration. The 72h-EbC50 (green algae, biomass) was > 50.0 mg/L and the 72h-NOECb was > 50.0 mg/L; the 72hErC50 (green algae, growth rate) was > 50.0 mg/L and the 72h-NOECr was 25.0 mg/L.

The validity criteria of the test guideline are fulfilled (OECD 201 adopted 1984) and results of the study are considered reliable. With regard to the validity criteria of the updated guideline (2006), the coefficients of variation for the section-by-section-growth rates of the controls and average specific growth rates were newly calculated (as far as available data allowed the assessment) and are below the permitted maximum value. Therefore, the study also meets the validity criteria of the updated guideline.

Description of key information

No aquatic toxicity data on plants for the submission substance is available. However adequate and reliable data for structural analogues is available (i.e. C8TM and C8C10TM, for read-across justification please see read-across report in section 13). The derived 72h-NOECr (green algae, growth rate) = 27.0 mg/L (corrected for the molecular weight of the target substance).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

27 mg/L

Additional information

No aquatic toxicity data for the submission substance is available. However adequate and reliable data for structural analogues (i.e. C8TM and C8C10TM, for read-across justification please see read-across report in section 13) are reported below.

In a reliable long-term toxicity study on green algae (according to OECD 201 and in compliance with GLP, MoE Japan, 2001, RL1), effects on the growth rate were observed after 72 hours of exposure, but not on yield of biomass (analogue substance: trioctyl benzene-1,2,4-tricarboxylate, trioctyl ester, C8TM, CAS no. 89-04-3). The tested concentrations lay above the water solubility of the test substance, except for the lowest of measured 0.79 mg/L (solubilizer was used). No significant adverse effects were observed in concentrations up to 25 mg/L (corresponding to 27 mg/L of the target substance). Test concentrations of the supporting study lay below the water solubility (lowest at nominal 0.24 mg/L), and no effects were observed up to the highest test concentration of 3.5 mg/L (OECD 201, GLP, Huels AG, 1994, RL2; analogue substance: 1,2,4-benzenetricarboxylic acid, mixed decyl and octyl triesters, C8C10TM, CAS no. 90218-76-1; corresponding to 3.3 -3.8 mg/L of the target substance). In conclusion, no toxicity to green algae is expected below the water solubility of the analogue and submission substance.