3,4,5-trifluorophenol

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:

from 2004-05-06 to 2004-02-03

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:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples were taken from all concentrations (10, 18, 32, 56 and 100 mg/L).
- Sampling method: The samples were analyzed 72 hours after start of the experimental part. Samples were used undiluted for HPLC-analysis
- Sample storage conditions before analysis: The samples were stored under the same conditions as the vessels with Algae and afterwards given to analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: An aliquot (about 50 mg) of the test material was weighed, dissolved with eluent and filled up with eluent to 50 mL.Appropriate amounts of the stock solution were diluted with eluent to get seven standard solutions in the range from 5 - 126 mg/L.
- Controls: blank control (performed with test solution without test substance), positive control performed with potassium dichromate.
- Evidence of undissolved material: No

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: algae
- Strain: No. SAG 86.81
- Source: Collection of Algae Cultures, Institute of Plant Physiology of the University of Gottingen
- Method of cultivation: The algae have been cultivated in the laboratories of Merck KGaA in Darmstadt (Germany) under standardized conditions.

ACCLIMATION
- Culturing media and conditions: same as the test
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

none

Post exposure observation period:

none

Hardness:

no data

Test temperature:

24 - 25 °C

pH:

pH without algae
Control: 8.02 (start) - 8.13 (after 72 hours)
Test concentrations: 7.38 - 7.84

pH with algae:
Control. 8.02 (start) - 10.46 (after 72 hours)
Test concentrations: from 7.38 (start, 100 mg/L) 10.46 (after 72 hours)

Dissolved oxygen:

n.a

Salinity:

n.a

Conductivity:

n.a

Nominal and measured concentrations:

- Nominal concentration: 0 (control), 10, 18, 32, 56 and 100 mg/L
- Measured concentration: 9.5, 17.28, 30.72, 56 and 100 mg/L. The analytically determined concentrations were between 94-101 % at the start and 95-100 % at the end, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed
- Fill volume: 120 mL
- Aeration: yes
- Initial cells density: 10,000 cells/mL
- Control end cells density: 673,234 cell/mL after 72 hours.
- No. of organisms per vessel: 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 2

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water according to OECD 201 (1984)
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH-values were measured in the control and all test material concentrations at the start (0 hours) and at the end (72 hours) of the experimental part. During the experimental part, the temperature was registered in a separate control flask with an electronic thermometer containing a maximum and minimum memory display.

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: continuously illumination.
- Light intensity: The light intensity was approx. 7500 to 9500 Lux.

EFFECT PARAMETERS MEASURED: Inhibitory effect of the test substance on the growth of the freshwater green algae species after 24, 48 and 72 hours of exposure in the test media.
- Determination of cell concentrations: The algae cell densities were determined with an electronic particle counter (Coulter, Z2).
- Chlorophyll measurement: Not determined

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: Yes. In a pretest under closed static conditions in 100 mL flasks, an inhibition of the growth rate of the algae was observed at the concentration of 100 mg/L nominally.
- Test concentrations: 0 (control), 100 mg/L
- Results used to determine the conditions for the definitive study: In the preculture, the factor of cell growth was approx. 162.7. The algae increased from 10,000 cells/mL at the start to 1,626,929 cells/mL after 72 hours.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

18.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.L. (16.6 - 20.5) mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

12.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 95 % C.L. (11.6 - 13.7 mg/L)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: not observed
- Unusual cell shape: not observed
- Colour differences: not observed
- Flocculation: not observed
- Adherence to test vessels: not observed
- Aggregation of algal cells: not observed
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
- Toxicity results: The 72h-EbC50 was determined to be 12.6 mg/L, the 72h-ErC50 was determined to be 18.4 mg/L and the 72 h-NOEC was determined to be < 10 mg/L.

Results with reference substance (positive control):

Regularly, a positive control test to check the test system is carried out with potassium dichromate Art. 104864 (HEUSENER, 2004). The results of this regular test show that the test meets the valided criteria.

Reported statistics and error estimates:

The average growth rate at each test material concentration was then compared with the control value and the percentage reduction in growth rate was calculated according to the procedure of UNKELBACH and WOLF (1985) using the PC-program 511.

Table 1: Nominal test material concentrations and the growth inhibition data after 72 hours exposure

Test material [mg/L]	Cell density per mL
Control		0 h	24 h	48 h	72 h
		10,000	67,293	305,031	646,391
		10,000	67,590	315,180	679,660
		10,000	68,130	292,009	693,651
	mean	10,000	67,671	304,073	673,234
10		10,000	44,397	190,689	585,990
		10,000	55,917	231,880	701,250
		10,000	51,957	173,961	656,931
	mean	10,000	50,757	198,843	648,057
18		10,000	25,263	36,900	43,803
		10,000	26,667	35,127	40,230
		10,000	24,390	24,623	38,070
	mean	10,000	25,440	35,550	40,701
32		10,000	16,650	16,677	16,677
		10,000	16,983	18,000	15,363
		10,000	16,470	15,417	15,993
	mean	10,000	16,701	16,698	16,011
56		10,000	15,967	17,100	14,283
		10,000	14,940	17,190	15,147
		10,000	15,480	14,697	13,653
	mean	10,000	15,459	16,329	14,361
100		10,000	16,560	16,110	16,047
		10,000	15,903	16,407	15,507
		10,000	17,667	18,567	17,163
	mean	10,000	16,710	17,028	16,239

 

Table 2: Nominal test material concentrations and the growth inhibition data in % after 72 hours exposure

Test material [mg/L]	Inhibition of cell growth in percent			Growth inhibition (0-72 hours)
	24 h	48 h	72 h	Ia	It
10	29.3	34.7	13.6	19.7	0.9
18	73.2	88.3	94.1	91.8	66.7
32	88.4	96.2	98.7	97.6	88.8
56	90.5	96.6	98.9	98.0	91.4
100	88.4	96.1	98.6	97.5	88.5

 

Ia = inhibition of cell growth in percent

lt = inhibition of growth rate in percent

 

Table 3: Nominal and corresponding analytical concentrations

Nominal concentration [mg/L]	Analytical concentration in % during the study
	start	72 h
10	94	95
18	94	96
32	98	96
56	98	100
100	101	100

 

 Table 4: pH Value

Concentration [mg/L]	pH without algae		pH with algae
	start	72 h	Start	72 h
Control	8.02	8.13	8.02	10.46
10	7.84	7.80	7.84	10.40
18	7.76	7.67	7.76	7.98
32	7.61	7.56	7.61	7.64
56	7.45	7.41	7.45	7.56
100	7.38	7.39	7.38	7.41

 

Validity criteria fulfilled:

yes

Conclusions:

The test substance was tested in a closed static test system according to OECD 201 (1984). Under the given conditions of this study the 72 h ErC50 value was determined to be 18.4 mg/L.

Executive summary:

The influence of the test material on the growth and growth rate of the green algae species Desmodesmus subspicatus was tested according to OECD 201 (1984) under GLP conditions. Because of the test material had a pungent odor in the chosen concentrations, 100 mL Erlenmeyer flasks were filled with 120 mL test medium and sealed with ground stoppers to form a closed system. The algae were exposed to different concentrations of test material and the growth was calculated after 24, 48, or 72 hours of exposure in the test media. Based on the results of a pretest, the study was performed with nominal concentrations of 10,18, 32, 56, and 100 mg test material/L reconstituted water. The study design included three replicates per test material concentration, each containing about 120 mL test medium and 10,000 cells/mL at the start of the experimental phase and a control without algae. Analyses of the test material concentrations were performed with all doses at the start and after 72 hours. The limit of quantification of the analytical method was 0.2 mg/L. The analytically determined concentrations were between 94 - 101 % at the start and 95 - 100 % at the end, respectively. During the experimental phase of the study, the test material concentrations could be maintained at > 80 % of the initial concentrations. Therefore, the EC50 values had to be calculated with the nominal concentrations. The median effective concentration (EC50), which would result in a 50 % growth inhibition and the 95% confidence interval compared to the control, was calculated from the nominal concentrations of the test substance. The 72h-ErC50 was determined to be 18.4 mg/L and the 72 h-NOEC was determined to be < 10 mg/L.

Description of key information

The test substance was tested to Desmodesmus subspicatus in a closed static test system according to OECD 201 (1984). Under the given conditions of this study (reference 6.1.5-1) the 72 h ErC50 value was determined to be 18.4 mg/L and the NOEC was determined to be < 10 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

18.4 mg/L

Additional information

The influence of the test material on the growth and growth rate of the green algae species Desmodesmus subspicatus was tested according to OECD 201 (1984) under GLP conditions. Because of the test material had a pungent odor in the chosen concentrations, 100 mL Erlenmeyer flasks were filled with 120 mL test medium and sealed with ground stoppers to form a closed system. The algae were exposed to different concentrations of test material and the growth was calculated after 24, 48, or 72 hours of exposure in the test media. Based on the results of a pretest, the study was performed with nominal concentrations of 10,18, 32, 56 and 100 mg test material/L reconstituted water. The study design included three replicates per test material concentration, each containing about 120 mL test medium and 10,000 cells/mL at the start of the experimental phase and a control without algae. Analyses of the test material concentrations were performed with all doses at the start and after 72 hours. The limit of quantification of the analytical method was 0.2 mg/L. The analytically determined concentrations were between 94 - 101 % at the start and 95 - 100 % at the end, respectively. During the experimental phase of the study, the test material concentrations could be maintained at > 80 % of the initial concentrations. Therefore, the EC50 values had to be calculated with the nominal concentrations. The median effective concentration (EC50), which would result in a 50 % growth inhibition and the 95 % confidence interval compared to the control, was calculated from the nominal concentrations of the test substance. The 72h-ErC50 was determined to be 18.4 mg/L and the 72 h-NOEC was determined to be < 10 mg/L.