1-[2-(2-chloroethoxy)phenylsulfonyl]-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)urea;triasulfuron (ISO)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

effects on growth of an additional algal species

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Nov 1985 to 04 Dec 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

Marine diatoms were exposed to the test substance under static conditions for 12 days. Mean maximum standing crop values were recorded to determine the EbC50 and the NOEbC of the test item to the diatoms.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were analyzed on day 0 and at the end of the assay. Samples for day 0 analyses were portions of the test treatments prepared to begin the assay. Day 12 samples were obtained from the "blanks" incubated for this purpose.
Samples were shipped on ice via an overnight delivery service to the analytical facility and were kept under refrigeration prior to analysis.

Vehicle:

yes

Remarks:

N,N-dimethylformamide

Details on test solutions:

To begin the definitive test, a stock solution of 200 mg/mL in DMF was prepared by weighing 5.1813 g of test substance, to the nearest 0.1 mg, and diluting to 25 mL in a volumetric flask with reagent-grade DMF. The test material was dissolved by 10 minutes of sonication in a water bath ultrasonic cleaning machine. A 100 mg/mL stock solution and a 50 mg/mL stock solution were prepared from the 200 mg/mL stock. Stock solutions were prepared on the day the assay was begun. Test concentrations were prepared by adding the required volumes of the appropriate stock solution to marine algal assay medium in 500 mL volumetric flasks. A solvent control treatment was prepared to contain an amount of DMF equivalent to the greatest amount of DMF present in any test material treatment. The amount of DMF in the solvent control was 0.5 mL/L. After thorough mixing, 50 mL of each concentration was added to each of four replicate test vessels. The control contained medium only, 50 mL in each of four replicate flasks. The four replicate flasks for each treatment were inoculated with algae. In addition, approximately 150 mL of each treatment, control and solvent control was placed in a fifth test vessel to serve as a blank to be used for the analytical determination of test concentrations at the end of the assay. The blanks were incubated with the inoculated flasks. Approximately 150 mL of each treatment, control and solvent control was retained for analysis of initial test concentrations.

Test organisms (species):

Skeletonema costatum

Details on test organisms:

TEST ORGANISM
- Source: Skeletonema costatum used in this test came from laboratory stock cultures. The original culture was obtained from the Culture Collection of Marine Phytoplankton at the Bigelow Laboratory for Ocean Sc iences, West Boothbay Harbor, Maine.
- Condition of cultivation: Stock cultures are maintained in synthetic marine algal assay medium in Erlenmeyer flasks under illumination of approximately 400 foot-candles (4304 lumens/m2), with a photoperiod of 14L:10D and at a temperature of 20 ± 2°C. Flasks are manually shaken once or twice daily (except on weekends). Transfers are made regularly into fresh medium to provide nine to twelve day old cultures for assay inoculations.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

12 d

Test temperature:

20 ± 2 °C

Salinity:

30 ‰

Nominal and measured concentrations:

- Nominal concentrations: 0 (blank control), 0 (solvent control), 10, 18, 32, 56 and 100 mg/L.
- Measured concentrations (day 0): < 2.5, < 2.5, 9.3, 11, 32, 65, 93 mg/L, respectively.
- Measured concentrations (day 12): < 2.5, < 2.5, 12, 16, 36.5, 63, 93 mg/L, respectively.
See Table 1 in 'Any other information on materials and methods incl. tables'.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type: Closed with foam stoppers to permit gas exchange
- Fill volume: 50 mL
- Initial cells density: 10,000 cells/mL
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- No. of vessels per vehicle control: 4

GROWTH MEDIUM
Synthetic sea water was filter-sterilized by passing through a 0.22 micron porosity membrane filter into a sterile container. Nutrients were added to the sterile synthetic sea water. For stock culture medium, 30 mL metal mix, 20 mL minor salt mix and 1.0 mL vitamin mix were added to 1 litre (final volume) of synthetic sea water.

TEST MEDIUM
To prepare medium for toxicity tests, 15 mL metal mix (without EDTA), 10 mL minor salt mix and 0.5 mL vitamin mix were added to 1 litre (final volume) of synthetic sea water. Medium was stored in the dark at 4 °C, and brought to room temperature prior to use.

OTHER TEST CONDITIONS
- Photoperiod: 14 hours light:10 hours dark
- Light intensity and quality: 4304 ± 650 lumens/m2 provided by overhead cool-white fluorescent lights
- Salinity (for marine algae): 30 ‰

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Coulter counter (particle counter)
- Frequency: Cell counts were made on test days 3, 4, 5, 7, 10, and 12.
- Other assessments: Dry weight was determined at the end of the test.

RANGE-FINDING TEST
A range-finding test conducted using six test concentrations from 0.001 to 100 mg/L indicated that concentrations of 10 to 100 mg/L would be appropriate for definitive testing.

Reference substance (positive control):

no

Key result

Duration:

12 d

Dose descriptor:

EC50

Effect conc.:

31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

12 d

Dose descriptor:

NOEC

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

An overview of the results is provided in Table 2 – Table 4 in ‘Any other information on results incl. tables’.
From the shapes of the growth curves, it is evident that increasing concentrations of test material were increasingly inhibitory to S. costatum. The lag phase of growth was lengthened by exposure to 32, 56 and 100 mg/L. Growth was totally inhibited by exposure to 100 mg/L and markedly reduced by exposure to 56 mg/L. Growth was slowed by exposure to 32 mg/L, although final population density in this test concentration approached that in the solvent control. Growth in cultures exposed to 10 and 18 mg/L was very similar to that in the solvent control. For each counting day, mean growth in each treatment was expressed relative to the control (Table 2) and solvent control (Table 3). Percent inhibition increases at increasing test concentrations, and in general, decreases over time. 

Five days has been suggested as the length for a short-term toxicity screening test and as the "exposure period" in an assay to determine algicidal and algistatic concentrations (Payne and Hall, 1979). Individual t-tests of the day 5 cell counts showed that the population density in the 18, 32, 56 and 100 mg/L test concentrations was significantly less than that in the solvent control. There were no significant differences between the day 5 cell counts in the control or 10 mg/L test concentration and solvent control. Effects of the test material on day 5 ranged from 9.5 % inhibition (10 mg/L) to 98.7 % inhibition (100 mg/L). Maximum standing crop (MSC), expressed as cells/mL, occurred by day 7 or 10 in the control, solvent control, and three lowest test concentrations. The assay was terminated on day 12, by which time populations in all flasks had reached MSC. ANOVA and Duncan's test indicated that the population density in the 32, 56 and 100 mg/L test concentration were significantly less than that in the solvent control. In none of the other test concentrations or in the control were the mean MSC's significantly different from that in the solvent control. Dry weight in mg/L may also be considered as an expression of maximum standing crop. ANOVA and Duncan's test indicated that the mean dry weights in the 56 and 100 mg/L test concentrations were significantly less than that in the solvent control. There were no significant differences between the mean dry weights in any of the other test concentrations and that in the solvent control. The percent effect of the test material, relative to the solvent control, based upon MSC in cells/mL, ranged from 13.8 % stimulation (control) to 98.4 % inhibition (100 mg/L). Based upon MSC in mg/L, percent effects ranged from 17.4 % stimulation (18 mg/L) to 21.91 inhibition (100 mg/L). It appears that MSC based on cells/mL is more reliable than the dry weight values due to improved accuracy of the cell count procedure over the dry weight procedure. 

EC values are given in Table 4. The NOEC, defined as the highest concentration tested that had no significant effect upon maximum standing crop (cells/mL or dry weight, (mg/L), relative to the solvent control, is 18 mg/L.

Reported statistics and error estimates:

The NOEC is indicated by the results of ANOVA and Duncan's test. For maximum standing crop expressed as cells/mL, the percent inhibition (relative to the solvent control) was plotted against concentration to determine the EC values.

 

Table 2. Percent inhibition*, relative to control, based upon cell counts during 12-day exposure period

Nominal Conc. mg/L		Day 3	Day 4	Day 5	Day 7	Day 10	Day 12
Solvent		0.8	4.4	6.1	12.2	8.8	9.9
10		7.8	12.2	15.0	21.9	11. 3	10.2
18		16.3	22.6	31.2	27.4	2.7	-2.5
32		59.7	67.4	68.4	60.8	24.8	21.0
56		86.0	93.0	96.0	97.0	93.9	90.2
100		91.5	96.7	98.8	99.4	99.2	99.1

 

*A negative percent inhibition indicates stimulation.

Table 3. Percent inhibition, relative to solvent control, based upon cell counts during 12-day exposure period

Nominal Conc mg/L	Day 3	Day 4	Day 5	Day 7	Day 10	Day 12
10	7.0	8.1	9.5	11.1	2.7	0.3
18	15.6	19.0	26.7	17.4	-6.7	-13.8
32	59.4	65.9	66.4	55.4	17.5	12.3
56	85.9	92.6	95.7	96.6	93.3	89.2
100	91.4	96.5	98.7	99.3	99.1	99.0

 

*A negative percent inhibition indicates stimulation.

Table 4. EC values

 

Based upon MSC, cells/mL
EC10:	14 mg/L
EC50:	31 mg/L
EC90:	67 mg/L
EC95:	85 mg/L

Deviations

The inoculum of algae used- to start the test was from a 7-day old stock culture rather than a 9 to 12-day old stock culture as specified in the protocol. This deviation is judged not to have affected the results of the study.

Validity criteria fulfilled:

not specified

Conclusions:

In a 12-d toxicity study on Skeletonema costatum, not conducted according to a standard guideline, the EbC50 was determined to be 31 mg/L and the NOEbC was 18 mg/L, based on nominal concentrations.

Executive summary:

The toxicity of the test item to marine diatoms (Skeletonema costatum) was determined in a static system and was in compliance with GLP criteria. The study was not conducted according to a standard guideline. Algae were exposed to nominal concentrations of 10, 18, 32, 56 and 100 mg/L, alongside a blank control and a solvent control (DMF) for 12 days. The measured concentrations in the test treatments were 9.3, 11, 32, 65 and 93 mg/L on day 0 and 12, 16, 36.5, 63 and 93 mg/L on day 12. Percent recovery ranged from 86 % to 109 %. The initial cell density was 10,000 cells/mL. The test conditions were: Temperature 20 ± 2 ˚C. A daily photoperiod of 14 hours with 4304 ± 650 lumens/m2. pH and microscopical observations of abnormalities were not reported.

The growth curves indicate that increasing concentrations of the test substance were increasingly inhibitory to S. costatum. The final population density was markedly reduced by exposure to 56 mg/L and growth was completely inhibited by exposure to 100 mg/L. Based upon cell counts on day 5, effects of the test material on algal growth ranged from 9.5 % inhibition (10 mg/L) to 96.5 % inhibition (100 mg/L), relative to the solvent control. By day 12, effects ranged from 13.8 % stimulation (18 mg/L) to 99.0 % inhibition (100 mg/L), relative to the solvent control. Mean maximum standing crop (MSC) values were expressed both as cells/mL and as mg/L dry weight. The mean MSC's (cells/mL) in the 32, 56 and 100 mg/L test concentrations were significantly less than that in the solvent control. The mean MSC's (mg/L dry weight) in the 56 and 100 mg/L test concentrations were significantly less than that in the solvent control. Based on nominal concentrations, the 12-day EbC50 was 31 mg/L. The NOEbC was 18 mg/L. 

Reference 2

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Nov 2010 to 30 Nov 2010

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:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

1996, Public Draft

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the control. For sampling at the end of the test, the test medium of the treatment replicates was pooled.
All samples were deep-frozen immediately after sampling and stored at about -20 °C until analysis. The concentrations of the test item were determined in one test medium sample per test concentration from both sampling dates (0 and 96 hours). From the control samples, one of the duplicate samples was analyzed from the corresponding sampling times. The algae were separated from the samples by filtration prior to analysis.

Vehicle:

no

Details on test solutions:

A dispersion with the loading rate of 5 mg/L was prepared at the start of the test by dissolving 15.02 mg of the test item in 3000 mL of test water. This preparation was supported by ultrasonic treatment for 30 minutes and intense stirring on a magnetic stirrer over 1 hour in the dark, to dissolve a maximum amount of the test item in the dispersion.
The stirring period of 1 hour was chosen according to the results of a pre-experiment (without GLP), which showed that the maximum concentration was reached after this time.
After the stirring period, the dispersion of the test item was filtered through a membrane filter (pore size 0.45 µm). The undiluted filtrate was used as a stock solution for preparation of the test media with lower test concentrations. For this preparation, the filtrate was serially diluted with test water. The test media were prepared just before the start of the test (= start of exposure).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: No. 61.81 SAG
- Source: SAG, Institute for Plant Physiology, University of Göttingen
- Method of cultivation: The algae are cultured in the test facility laboratories under standardized conditions according to the test guidelines.

ACCLIMATION
An inoculum culture was set up three days before the start of the exposure. The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

24 mg/L as CaCO3

Test temperature:

22 °C

pH:

- Test start: 8.0
- Test end: 8.1 to 8.8

Nominal and measured concentrations:

- Nominal concentrations: 0 (blank control), 0.023, 0.050, 0.11, 0.23, 0.50 and 1.1 mg/L
- Measured concentrations day 0: < LOQ, 0.020, 0.0435, 0.0951, 0.194, 0.447 and 0.949 mg/L, respectively.
- Measured concentrations day 4: < LOQ, 0.0192, 0.0431, 0.0927, 0.196, 0.443 and 0.946 mg/L, respectively.
See Table 1 in 'Any other information on materials and methods incl. tables'.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks containing 15 mL of test solution.
- Type: Closed (covered with a glass dish)
- Mixing condition: During the test, the test solutions were continuously stirred by magnetic stirrers.
- Initial cells density: 5000 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: Yes

WATER PARAMETERS
- Intervals of water quality measurement: The pH was measured and recorded in each test concentration and the control at the start and at the end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test media was also recorded daily. Additionally, the water temperature was continuously recorded with a data logger.

OTHER TEST CONDITIONS
- Photoperiod: Continuous
- Light intensity: Approximately 5200 lux (range: 4520 to 5680 lux)
- Light source: fluorescent tubes (36W/840), installed above the test flasks; The light intensity over the incubation area (measured at nine places in the experimental area) was within a ±15%-deviation from the average light intensity.

EFFECT PARAMETERS MEASURED
- Sampling intervals: A small volume of the algal suspension was withdrawn from each test flask daily for the measurement of the biomass, and was not replaced.
- Determination of cell concentrations: The algal biomass in the samples was determined by fluorescence measurement The measurements were performed at least in duplicate.
- Determination of cell shape: At the end of the test, a sample was taken from the control and from the test concentration of nominal 0.23 mg/L. The shape and size of the algal cells were examined microscopically in these samples. This test concentration was chosen, since at the higher nominal concentrations, the algal cell density was too low for a reliable examination.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.39 mg/L

95% CI:

>= 0.37 - <= 0.42

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.033 mg/L

95% CI:

>= 0.028 - <= 0.038

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

yield and biomass

Remarks on result:

not determinable

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.054 mg/L

95% CI:

>= 0.05 - <= 0.058

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.011 mg/L

95% CI:

>= 0.009 - <= 0.013

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.063 mg/L

95% CI:

>= 0.058 - <= 0.068

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Area under curve

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.012 mg/L

95% CI:

>= 0.009 - <= 0.014

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Area under curve

Details on results:

An overview of the results is provided in Table 2 – Table 5 in ‘Any other information on results incl. tables’.
- Inhibition of the algae growth: The mean cell density, given as relative fluorescence units, after 72h exposure was 1.42E+05, 1.09E+05, 0.75E+05, 0.38E+05, 0.20E+05, 0.11E+05, 0.05E+05 in blank control, 0.023, 0.050, 0.11, 0.23, 0.50 and 1.1 mg/L treatments, respectively. When compared to the cell density in the blank control, the 0.023, 0.050, 0.11, 0.23, 0.50 and 1.1 mg/L treatments showed a growth rate inhibition of 5.5, 13.1, 27.4, 41.0, 52.7 and 70.5%, respectively, after 72 hours of exposure.
The test item had a significant inhibitory effect on the growth of the algae (AUC and yield) after the test periods of 72 and 96 hours at the concentration of 0.023 mg/L and at all higher test concentrations. Thus, this concentration was determined to be the 72- and 96-hour LOEC. The 72- and 96-hour NOEC could not be determined since up to and including the test concentration of 0.023 mg/L the AUC and yield of the algae after 72 and 96 hours were significantly lower than in the control. The 96-hour NOEC for growth rate was 0.023 mg/L since at this test concentration no significant inhibitory effect could be determined.
- Cell size and shape: The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 0.23 mg/L and the algal cells in the control. The shape and size of the algal cells was obviously not affected by the test item up to at least this concentration.
- Other observations: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

Validity criteria
In the control the biomass increased by a factor of 127 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 and 96 hours was 3.2 and 33%, respectively. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control after 72 and 96 hours was 0.5%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

Results with reference substance (positive control):

The report of the test facility gives a 72 hour EC50 value for growth rate of 0.99 mg/L, which is within the test facility's historical range of 0.71 to 1.7 mg/L.

Reported statistics and error estimates:

The EC values and their 95% confidence intervals were calculated by Probit Analysis.

The area under the curve, average growth rate and yield at the test concentrations were compared to the control values by Dunnett t-test in order to obtain the NOEC.

Table 2 Area under the growth curve (AUC) and inhibition of AUC

Nominal test item Concentration (mg/L)	Areas under the growth curves AUC (1.0E+03*day) and inhibition of AUC (IAUC)
	0-24 h		0-48 h		0-72 h		0-96 h
	AUC	IAUC (%)	AUC	IAUC (%)	AUC	IAUC (%)	AUC	IAUC (%)
Control	2.2	0.0	18.2	0.0	102.5	0.0	315.4	0.0
0.023	2.1	5.5	15.5*	14.6	80.8*	21.1	266.2*	15.6
0.050	1.7*	21.8	12.7*	30.1	59.1*	42.3	221.7*	29.7
0.11	1.2*	43.6	7.5*	58.6	30.9*	69.9	107.8*	65.8
0.23	0.9*	60.9	4.8*	73.8	17.0*	83.4	52.9*	83.2
0.50	0.6*	73.6	3.0*	83.5	9.8*	90.4	26.2*	91.7
1.1	0.2*	91.2	1.0*	94.7	3.3*	96.8	8.0*	97.5

*: mean value significantly lower than in the control (according to Dunnett t test, one sided, α = 0.05)

Table 3 Average growth rate and inhibition of growth rate

Nominal test item Concentration (mg/L)	Average growth rate µ (1/day) and inhibition of µ (Ir)
	0-24 h		0-48 h		0-72 h		0-96 h
	µ	Ir (%)	µ	Ir (%)	µ	Ir (%)	µ	Ir (%)
Control	1.60	0.0	1.62	0.0	1.62	0.0	1.39	0.0
0.023	1.55	2.8	1.53*	5.7	1.53*	5.5	1.37*	1.4
0.050	1.41*	12.0	1.43*	11.9	1.40*	13.1	1.35*	2.3
0.11	1.17*	26.7	1.15*	29.0	1.17*	27.4	1.17*	15.9
0.23	0.93*	41.7	0.93*	42.5	0.95*	41.0	0.97*	30.0
0.50	0.71*	55.4	0.73*	55.2	0.76*	52.7	0.77*	44.8
1.1	0.30*	81.4	0.36*	78.1	0.48*	70.5	0.46*	67.1

*: mean value significantly lower than in the control (according to Dunnett t test, one sided, α = 0.05)

Table 4 Yield and inhibition of yield

Nominal test item Concentration (mg/L)	Yield Y (x10^3) and inhibition of Y (Iy)
	0-24 h		0-48 h		0-72 h		0-96 h
	Y	Iy (%)	Y	Iy (%)	Y	Iy (%)	Y	Iy (%)
Control	4.4	0.0	27.6	0.0	140.9	0.0	284.9	0.0
0.023	4.2	5.5	22.8*	17.5	107.8*	23.5	262.9*	7.7
0.050	3.4*	21.8	18.6*	32.7	74.2*	47.3	250.9*	11.9
0.11	2.5*	43.6	10.1*	63.5	36.6*	74.0	117.3*	58.8
0.23	1.7*	60.9	6.1*	77.9	18.3*	87.0	53.4*	81.2
0.50	1.2*	73.6	3.7*	86.7	10.0*	92.9	22.8*	92.0
1.1	0.4*	91.2	1.2*	95.8	3.6*	97.5	5.8*	98.0

*: mean value significantly lower than in the control (according to Dunnett t test, one sided, α = 0.05)

Table 5 Effect parameters

	after 72 h			after 96 h
Parameter (mg/L)	AUC	Growth rate	Yield	AUC	Growth rate	Yield
EC50 95% CI	0.063 0.058–0.068	0.39 0.37–0.42	0.054 0.050–0.058	0.079 0.073–0.085	0.57 0.53–0.61	0.10 0.094–0.11
EC20 95% CI	0.021 0.018–0.024	0.077 0.069–0.085	0.019 0.017–0.021	0.031 0.027–0.034	0.16 0.14–0.17	0.052 0.044–0.060
EC10 95% CI	0.012 0.009–0.014	0.033 0.028–0.038	0.011 0.009–0.013	0.019 0.016–0.022	0.079 0.068–0.090	0.036 0.029–0.043
NOEC	n.d.	n.d.	n.d.	n.d.	0.023	n.d.
LOEC	0.023	0.023	0.023	0.023	0.050	0.023

95% CI: 95% confidence interval

n.d.: could not be determined

Validity criteria fulfilled:

yes

Remarks:

See ‘Details on results’

Conclusions:

In a toxicity study on green algae following OECD TG 201, OPPTS 850.5400 and EU Method C3 guidelines, the 72-h ErC50 and ErC10 values were determined to be 0.39 and 0.033 mg/L, respectively. The NOEC value could not be determined.

Executive summary:

This study was conducted to determine the toxicity of the test item on green algae (Raphidocelis subcapitata) according to OECD TG 201, OPPTS 850.5400 and EU Method C3 test guidelines, and in compliance with GLP principles. The algae were exposed for 96 hours in a static system at nominal test concentrations of 0 (blank control), 0.023, 0.050, 0.11, 0.23, 0.50 and 1.1 mg/L with an initial cell density of 5000 cells/mL (corresponding to 1.12E+03 relative fluorescence units). The test concentrations were analysed at 0 and 96 hours by using LC-MS/MS-detection. The measured concentrations were between 85 and 89% of the nominal values at the start and between 83 and 89% at the end of the test. The test was carried out under the following conditions: temperature 22 °C, continuous light (4520 - 5680 Lux), pH 8.0 at the start and pH 8.1 – 8.8 at the end of the test. The EC values were calculated by Probit analysis and the NOEC values were determined by using the Dunnett t-test. 

The mean cell density, given as relative fluorescence units, after 72h exposure was 1.42E+05, 1.09E+05, 0.75E+05, 0.38E+05, 0.20E+05, 0.11E+05, 0.05E+05 in blank control, 0.023, 0.050, 0.11, 0.23, 0.50 and 1.1 mg/L treatments, respectively. When compared to the cell density in the blank control, the 0.023, 0.050, 0.11, 0.23, 0.50 and 1.1 mg/L treatments showed a growth rate inhibition of 5.5, 13.1, 27.4, 41.0, 52.7 and 70.5%, respectively, after 72 hours of exposure. The mean cell density, given as relative fluorescence units, after 96h exposure was 2.86E+05, 2.64E+05, 2.52E+05, 1.18E+05, 0.55E+05, 0.24E+05, 0.07E+05 in blank control, 0.023, 0.050, 0.11, 0.23, 0.50 and 1.1 mg/L treatments, respectively. When compared to the cell density in the blank control, the 0.023, 0.050, 0.11, 0.23, 0.50 and 1.1 mg/L treatments showed a growth rate inhibition of 1.4, 2.3, 15.9, 30.0, 44.8 and 67.1%, respectively, after 96 hours of exposure. Based on nominal concentrations, the 72-hour ErC50 was 0.39 mg/L, the EyC50 was 0.054 mg/L and the EbC50 was 0.063 mg/L. The 96-hour ErC50 was 0.57 mg/L, the EyC50 was 0.10 mg/L and the EbC50 was 0.079 mg /L.

Description of key information

All available data was assessed and the studies representing the worst-case effects and/or relevant exposure periods are included here as key studies. Other studies are included as supporting information.

Freshwater, 72-h ErC10 = 0.033 mg/L (based on nominal concentrations), Raphidocelis subcapitata, OECD TG 201, OPPTS 850.5400 and EU Method C3, Liedtke 2011

Freshwater, 72-h ErC50 = 0.39 mg/L (based on nominal concentrations), Raphidocelis subcapitata, OECD TG 201, OPPTS 850.5400 and EU Method C3, Liedtke 2011

 

Marine water, 12-d NOEbC = 18 mg/L (based on nominal concentrations), Skeletonema costatum, other than guideline, Hughes 1986

Marine water, 12-d EbC50 = 31 mg/L (based on nominal concentrations), Skeletonema costatum, other than guideline, Hughes 1986

Key value for chemical safety assessment

EC50 for freshwater algae:

0.39 mg/L

EC50 for marine water algae:

31 mg/L

EC10 or NOEC for freshwater algae:

0.033 mg/L

EC10 or NOEC for marine water algae:

18 mg/L

Additional information

There are four studies available with freshwater algal species. All of them were performed under GLP but only two of them were performed according to standard guidelines. Of these two studies, the 72-h study (Liedtke 2011) on Raphidocelis subcapitata was selected as the key study because it represents the worst-case effects. The algae were exposed for 96 hours in a static system at nominal test concentrations of 0 (blank control), 0.023, 0.050, 0.11, 0.23, 0.50 and 1.1 mg/L with an initial cell density of 5000 cells/mL (corresponding to 1.12E+03 relative fluorescence units). The measured concentrations were between 85 and 89% of the nominal values at the start and between 83 and 89% at the end of the test. The test was carried out under the following conditions: temperature 22 °C, continuous light (4520 - 5680 Lux), pH 8.0 at the start and pH 8.1 – 8.8 at the end of the test. Based on nominal concentrations, the 72-hour ErC50 was 0.39 mg/L and the ErC10 was 0.033 mg/L.

One study with a marine water algal species is available for this endpoint, which was selected as key study. The toxicity of the test item to marine diatoms (Skeletonema costatum) was determined in a static system and was in compliance with GLP criteria. The study was not conducted according to a standard guideline. Algae were exposed to nominal concentrations of 10, 18, 32, 56 and 100 mg/L, alongside a blank control and a solvent control (DMF) for 12 days. The measured concentrations in the test treatments were 9.3, 11, 32, 65 and 93 mg/L on day 0 and 12, 16, 36.5, 63 and 93 mg/L on day 12. The initial cell density was 10,000 cells/mL. The test conditions were: Temperature 20 ± 2 ˚C. A daily photoperiod of 14 hours with 4304 ± 650 lumens/m2. pH and microscopical observations of abnormalities were not reported. Based on nominal concentrations, the 12-day EbC50 was 31 mg/L. The NOEbC was 18 mg/L.

The other three studies on different freshwater species are included as supporting studies. Anabaena flos-aquae was exposed to nominal concentrations of 0.20, 0.63, 2.0, 6.3 and 20 mg/L, alongside a culture medium control for 96 hours. The measured concentrations in the test media of the test concentrations of 0.20 to 20 mg/L were between 89 and 91% of the nominal values at the start of the test and between 86 and 95% at the end of the test. Based on nominal concentrations, the 72-hour ErC50 was 2.3 mg/L. The 72-hour NOErC was 0.20 mg/L (Liedtke 2011b). Navicula pelliculosa was exposed to nominal concentrations of 10, 18, 32, 56 and 100 mg/L, alongside a blank control and a solvent control (DMF) for 14 days. Analytical results showed that the initial concentrations on day 0 were in agreement with nominal (95 - 120 %). After 14 days of exposure the highest test concentration (nominal 100 mg/L) had decreased to 71 mg/L, while all lower concentrations were still in agreement with nominal (96 - 110 %). Although the recovery rate at 100 mg/L was < 80 % of nominal concentration the effect parameters were based on nominal concentrations. Based on nominal concentrations, the 14-day EbC50 was > 100 mg/L. The NOEbC was stated as < 100 mg/L (Hughes 1986a).

The study on Selenastrum capricornutum (Hughes 1985) was assessed as not reliable due to long exposure period and no analytical measurements were performed to determine the actual test concentrations in the test medium.