Trifluoroiodomethane

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:

02 Jun 2019 to 05 Jun 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

July 2011.

Qualifier:

according to guideline

Guideline:

other: Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals, OECD series on testing and assessment number 23, 2019.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Double samples for possible analysis were taken from all test concentrations and the control.
- Frequency: at t=0 h, t=24 h and t=72 h.
- Volume: 0.1 and 1.0 mL from the approximate centre of the test vessels; the 0.1 mL samples were directly diluted with 0.9 mL blank medium.
- Storage: samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Preparation of test solutions started with a minimum loading rate of 500 mg/L applying a 4 – 5 hour period of shaking in Tedlar® gas sampling bags to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle overnight (approximately 19 hours). Thereafter, the aqueous Saturated Solution (SS) was used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Freshwater algae
- Strain: NIVA CHL 1
- Source: In-house laboratory culture.

CULTURING CONDITIONS
- Stock culture: algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C
- Light intensity: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
- Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using tapwater purified by reverse osmosis;
- Pre-culture: 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1.0E+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Pre-culture medium: adjusted M2; according to the OECD 201 Guideline,

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

21.9 - 22.3°C

pH:

7.4 - 7.8

Nominal and measured concentrations:

Nominal concentrations - saturated solution (SS) prep. at a minimal loading rate of 500 mg/L: 1, 3.2, 10, 10, 32 and 100%
Measured concentrations - Time Weighted Average (TWA), respectively: 0.42, 1.3, 2.3, 2.9. 12 and 50 mg/L. See 'Any other information on materials and methods incl. tables'.

Details on test conditions:

TEST SYSTEM
- Test vessels: 40 mL, airtight closed with no headspace to prevent any loss of the test item due to volatilization.
- Cell density: an initial cell density of 1.0E+04 cells/mL.
- Illumination: Continuously using TLD-lamps with a light intensity within the range of 75 to 78 μE/m2/s.
- Incubation: vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
- Control: test medium without test item or other additives.

Replicates:
- 3 replicates of each test concentration,
- 6 replicates of the control,
- 1 extra replicate of each test group for sampling purposes after
- 24 hours of exposure,
- 1 or 2 replicates of each test concentration without algae.

WATER QUALITY MEASUREMENTS
- pH:At the beginning and at the end of the test, for all test concentrations and the control
- Temperature of medium: Continuously in a temperature control vessel.

EFFECT PARAMETERS MEASURED: cell density
- Cell density: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length =10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
- Cell appearance: At the end of the final test microscopic observations were performed on the 100% of the SS and the control to observe for any abnormal appearance of the algae.

RANGE FINDING STUDY
Three replicates of exponentially growing algal cultures were exposed to 1.0, 10 and 100% of the SS prepared at a minimum loading rate of 500 mg/L and to a control in a range-finding test. The final test was performed based on the results of a preceding range-finding test.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

46 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval ranging from 43 to 49 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

21 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

24 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence interval ranging from 17 to 35 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

13 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

MEASURED TEST SUBSTANCE CONCENTRATIONS
The measured concentrations at the start of the test were 0.11, 0.94, 0.31, 25 and 120 mg/L, respectively. During the exposure period, the concentrations decreased to 15 - 55% of initial at the end of the test, except for the 10% of the SS which increased to 535% of initial at the end of the test. A reason for this is unknown. The concentration measured in the 10% of the SS at the start of the test seems unreliable based on the results at t=24 h and at the end of the test. It is likely that the 10% of the SS is approximately the same as in the vessel without algae at the start of the test. Additionally, extra peaks were observed in the 1.0 and 10% of the SS at the start of the test. However, since biological effect was only observed at the highest test item, these measurements and observations will have no impact on the study conclusions. The concentrations measured in the samples taken from solutions with algae were comparable with the concentrations measured in the samples without algae at t=24 h and at the end of the test. Hence, it can be stated that the presence of the algae did not affect the concentration of the test item in test medium throughout the test. Based on these results, the average exposure concentrations were calculated and used to express effect parameters (See Table 1 in 'Any other information on materials and methods incl. tables)'.

INHIBITION OF GROWTH RATE AND INHIBITION OF YIELD
Table 2 shows group mean growth rates and the percentages of growth rate inhibition (total test period) whereas Table 3 shows the values at different time intervals. The group mean yields and the percentages of yield inhibition are summarized in Table 4. Statically significant growth rate and yield inhibition was only recorded at the highest test concentration of the test substance when compared to the control group, resulting in 56 and 94% inhibition, respectively. The NOEC based on statistical significance and biological relevance was thus set at 12 mg/L (32% of the SS). Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 50 mg/L when compared to the control.

Reported statistics and error estimates:

For determination of the NOEC and the ECx the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller) or inhibition of yield (Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm, α=0.05, one-sided, smaller). Calculation of ECx-values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test item. ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 2. Growth rate and percentage inhibition for the total test period

Test substance TWA conc. (mg/L)	Mean	Std. dev	n	% inhibition
Control	1.59	0.0147	6
0.42	1.616	0.0360	3	-1.7
1.3	1.598	0.0612	3	- 0.52
2.3	1.618	0.0217	3	- 1.8
12	1.573	0.0320	3	1
50	0.702	0.0730	3	56*

 

Table 3. Growth rate and percentage inhibition at different time intervals

Test substance TWA conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72 h
		Mean	% Inhibition	Mean	% Inhibition	Mean	% Inhibition
Control	6	1.503		1.519		1.747
0.42	3	1.512	- 0.6	1.537	- 1.2	1.8	- 3
1.3	3	1.327	12	1.692	- 11	1.774	- 1.5
2.3	3	1.528	- 1.7	1.571	- 3.4	1.756	- 0.52
12	3	1.388	7.6	1.542	- 1.6	1.790	- 2.4
50	3	1.235	18	- 0.037	102	0.908	48

 

Table 4. Yield and percentage inhibition for the total test period

Test substance TWA conc. (mg/L)	Mean	Std. dev	n	% inhibition
Control	116.9	5.25	6
0.42	127.1	14.21	3	- 8.7
1.3	121.1	23.43	3	- 3.6
2.3	127.6	8.24	3	- 9.2
12	111.5	11.06	3	4.6
50	7.4	1.86	3	94*

 

Table 5. Effect parameters

Parameter (mg/L)		NOEC	EC10	EC20	EC50
Growth rate	Value	12	21	27	46
	lower 95 % - cI		14	21	43
	upper 95 % - cI		30	35	49
Yield	Value	12	13	16	24
	lower 95 % - cI		8.3	11	17
	upper 95 % - cI		21	25	35

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, test substance inhibited growth rate and yield of this freshwater algae species significantly at TWA concentrations of 50 mg/L. The 72h-EC50 for growth rate inhibition (ErC50) was 46 mg/L with a 95% confidence interval ranging from 43 to 49 mg/L. The 72h-EC50 for yield inhibition (EyC50) was 24 mg/L with a 95% confidence interval ranging from 17 to 35 mg/L. The 72h-ErC10 was determined to be 21 mg/L and The 72h-EyC10 was determined to be 13 mg/L.

Executive summary:

The objective of the study was to evaluate the test substance for its ability to generate toxic effects in Raphidocelis subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10, EC20 and EC50 for both inhibition of growth rate and inhibition of yield. The study procedures were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2018. A Saturated Solution (SS) of the test substance was prepared at a minimal loading rate of 500 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium. A final test was performed based on the results of a preceding range-finding test. Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to solutions containing 1.0, 3.2, 10, 32 and 100% of the SS prepared at a minimal loading rate of 500 mg/L. The initial algal cell density was 1.0E+04 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure. The measured concentrations at the start of the test were 0.11, 0.94, 0.31, 25 and 120 mg/L, respectively. During the exposure period, the concentrations decreased to 15 - 55% of initial at the end of the test, except for the 10% of the SS which increased to 535% of initial at the end of the test. Since biological effect was only observed at the highest test item concentration, it will have no impact on the study conclusions. Based on these results, the average exposure concentrations were calculated and used to express effect parameters. Statically significant growth rate and yield inhibition was only recorded at the highest test concentration of the test substance when compared to the control group, resulting in 56 and 94% inhibition, respectively. The EC50 for the growth rate inhibition was 46 mg/L and the NOEC based on statistical significance and biological relevance was thus set at 12 mg/L (32% of the SS). The 72h-ErC10 was determined to be 21 mg/L and The 72h-EyC10 was determined to be 13 mg/L. The study was considered valid as all acceptance criteria were fulfilled.

Description of key information

The 72-h ErC50 and 72-h ErC10 were determined to be 46 and 21 mg/L, respectively in a GLP-compliant study in accordance with OECD TG 201 using Raphidocelis subcapitata.

Key value for chemical safety assessment

EC50 for freshwater algae:

46 mg/L

EC10 or NOEC for freshwater algae:

21 mg/L

Additional information

The objective of the study was to evaluate the test substance for its ability to generate toxic effects in Raphidocelis subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10, EC20 and EC50 for both inhibition of growth rate and inhibition of yield. The study procedures were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2018. A Saturated Solution (SS) of the test substance was prepared at a minimal loading rate of 500 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium. A final test was performed based on the results of a preceding range-finding test. Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to solutions containing 1.0, 3.2, 10, 32 and 100% of the SS prepared at a minimal loading rate of 500 mg/L. The initial algal cell density was 1.0E+04 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure. The measured concentrations at the start of the test were 0.11, 0.94, 0.31, 25 and 120 mg/L, respectively. During the exposure period, the concentrations decreased to 15 - 55% of initial at the end of the test, except for the 10% of the SS which increased to 535% of initial at the end of the test. Since biological effect was only observed at the highest test item concentration, it will have no impact on the study conclusions. Based on these results, the average exposure concentrations were calculated and used to express effect parameters. Statically significant growth rate and yield inhibition was only recorded at the highest test concentration of the test substance when compared to the control group, resulting in 56 and 94% inhibition, respectively. The EC50 for the growth rate inhibition was 46 mg/L and the NOEC based on statistical significance and biological relevance was thus set at 12 mg/L (32% of the SS).The 72h-ErC10 was determined to be 21 mg/L and The 72h-EyC10 was determined to be 13 mg/L. The study was considered valid as all acceptance criteria were fulfilled.

An additional computational estimation was provided as supporting information. The results are in line with the experimental study.

ECOSAR v2.00 data was generated on the test substance. Estimations of the test substance were conducted based on the model for neutral organics identified by ECOSAR. The 96-h EC50 and chronic value (ChV) were estimated to be 67.4 and 17.7 mg/L, respectively. The substance did not (completely) fall within the applicable domain of the model. However, it is expected that this does not affect the reliability of the prediction. Adequate and reliable documentation / justification of the ECOSAR prediction is provided.