tetradecylalcohol ethoxylate methyloxirane

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:

16 Nov 2018 - 30 Jan 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:

2006, Annex 5 corrected 2011

Deviations:

no

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

Version / remarks:

2nd edition, adopted July 6, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test concentrations and the control; stock solutions in ACN used for preparation of test solutions.
- Sampling method: 2.0 mL from the approximate centre of the test vessels at t=0 h, t=24 h and t=72 h
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
- At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling. For the WAF prepared at 0.10 mg/L, one of the replicates was sampled separately, since the algal cell density was considerably higher in this replicate. The other two replicates at this test concentration were pooled and subsequently also sampled.
- Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at the WAF prepared at a loading rate of 0.10 mg/L but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Vehicle:

yes

Remarks:

Acetonitrile

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method for Final Test:
Water Accommodated Fractions (WAFs) were prepared by using stock solutions in acetonitrile (ACN) at nominal concentrations of 0.10, 1.0, 3.2, 10, 32 and 100 mg/L. Empty test vessels were individually spiked with 0.5 mL of the respective stock. Each replicate of the control received 0.5 mL blank ACN. The solvent was completely evaporated overnight prior to addition of the test medium to avoid modification of WAF-composition due to presence of a water-miscible solvent. Thereafter, 50 mL test medium was added to each vessel. Test solutions, including those of the control, were agitated for a period of two days to ensure maximum dissolution of test item in test medium. Vessels were sealed during agitation to prevent vaporization of test medium. All test solutions were generally clear and colourless at the end of the preparation procedure. After preparation, 1 mL of an algal suspension was added to each replicate, providing a cell density of 10^4 cells/mL.
- Controls:Test medium without test item or other additives (blank control) and test medium without test item but with addition of the solvent used in the treatment of the stock solutions (solvent control).
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetonitrile

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum at test initiation: 3 days
- Method of cultivation: 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.
- Stock culture medium: M1 medium, according to NPR 6505, formulated using tap-water purified by reverse osmosis.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 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 and test medium: M2 medium, according to OECD 201

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

22 - 23°C

pH:

At start (t=0 h): 7.8 - 7.9
At end (t=72 h): 7.9 - 8.1

Nominal and measured concentrations:

Nominal: control, solvent control, and WAFs individually prepared at loading rates of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L
Measured: The measured concentrations at the start of the test were 6.3, 8.9, 49, 173 and 624 µg/L. During the exposure period, the initial concentrations in the WAFs prepared at 0.010 and 0.032 mg/L decreased below the LOD (0.31 µg/L). In the WAFs prepared at loading rates of 0.10 mg/L and higher, the concentrations were in the range of 16 to 73% of the initial concentrations at the end of the test. Time Weighted Average concentrations were calculated, see 'Any other information on materials and methods' for the calculation. See Table 1 in 'Any other information on results' for details on measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution
- Aeration: no
- Initial cells density: 10^4 cells/mL
- Control end cells density: 196 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Additional replicates: 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.

GROWTH MEDIUM
- Standard medium used: yes, M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2, formulated using tap-water purified by reverse osmosis.
- Intervals of water quality measurement: 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.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod and light intensity: Continuously using TLD-lamps with a light intensity within the range of 86 - 87 µE.m-2.s-1.
- Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: 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 immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
- Appearance of the cells: At the end of the final test, microscopic observations were performed on the WAF prepared at 0.032 mg/L to observe for any abnormal appearance of the algae compared to the blank control.

TEST CONCENTRATIONS
- Combined Limit/Range finding study test concentrations: untreated control and WSFs individually prepared at loading rates of 1.0, 10 and 100 mg/L. No solvent was used.
- Range-finding test: untreated control and WAFs individually prepared at loading rates in the range of 0.001 to 1.0 mg/L increasing by a factor of 10, using acetonitrile as solvent.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate, performed Jan 2019

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

25 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% CI: 19 - 35 µg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.3 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% CI: 1.6 - 5.4 µg/L

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 0.094 - 0.14 mg/L

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.033 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 0.019 - 0.045 mg/L

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.032 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Complete overview of effect concentrations based on both loading rate as well as TWA concentrations can be found in Table 2 and Table 3 in 'Any other information on results'. Measurements were based on the most abundant signal at m/z 420.3, which corresponds with the epoxide distribution. By using the analysed concentrations for expression of NOEC and ECx-values, it is assumed that this fraction is representative for the whole test item. However, it is unknown whether the analytically monitored constituent is responsible for the recorded effects. Therefore, these effect parameters should be interpreted with caution, since other test item constituents that are not analytically monitored might be responsible for the recorded effects as well.
- Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the WAF prepared at a loading rate of 0.032 mg/L when compared to the blank control.
- Data on algal growth obtained in the blank and solvent control did not significantly deviated from each other (p>0.05) and was therefore pooled.
- Growth rate inhibition increased with increasing loading rate from 0.010 mg/L upwards, resulting in 96% growth rate inhibition at the highest test concentration. Statistically significant growth rate inhibition was found at all test concentrations. However, inhibition of growth rate at loading rates of 0.010 and 0.032 mg/L was below 10% and therefore considered to be biologically not relevant. The NOELR based on biological relevance was thus 0.032 mg/L for the effects on growth rate.
It should be noted that the recorded cell density in one of the replicates prepared at a loading rate of 0.10 mg/L was considerably higher than in the other two replicates. Hence, an additional sample was taken from the deviant replicate at the end of the test. Analysis revealed a slightly lower concentration of 2.3 µg/L compared to 7.9 µg/L in the other replicates which were pooled prior to sampling. This might explain the difference in algal density between replicates. In order to prevent a bias, all data was included in the statistical analysis for determination of the effect parameters. It was considered that the variation between replicates had a negligible impact on the overall interpretation of the study results.
- Analytical results:No test item responses were detected in the samples taken from the blank and solvent control. The concentrations measured in the solution incubated without algae were comparable of those measured in its counterpart incubated with algae at the start of the test. The concentration in the solution without algae was however higher than in the solution with algae after 24 hours of exposure. The opposite was found at the end of the test, i.e. the concentration in the solution without algae was lower than in the solution incubated with algae. Hence, no conclusions can be drawn on effects of the algae on test item concentrations throughout the test.
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- The EC50 for growth rate inhibition (72h-ErC50) was 1.30 mg/L with a 95% confidence interval ranging from 1.29 - 1.32 mg/L. The 72h-ErC50 was within the expected range of 0.92 and 1.46 mg/L as specified in ISO
International Standard 8692, February 2012, and the historical range of 0.86 and 2.3 mg/L, which is based on reference tests performed at the Test Facility during the last ten years. In conclusion, the sensitivity of this culture of Raphidocelis subcapitata was in agreement with ISO International Standard 8692, February 2012 and the historical data collected at the Test Facility.

Reported statistics and error estimates:

A student t-Test (parametric, α=0.05, two-sided) was performed to test for presence of statistically significant differences in algal growth between the blank and solvent control. In case criteria for normal distribution of data and homogeneous variances were not met, a non-parametric Two-sample Mann-Whitney U-test (α=0.05, two-sided) was performed.
For determination of the effect parameters (i.e. NOELR, NOEC, ELx, and ECx-values) 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 controls (pooled data) revealed significant inhibition of growth rate (Step-down Jonckheere-Terpstra Test, α=0.05, one-sided, smaller).
Calculation of ELx-values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition versus the logarithms of the corresponding loading rates. ECx-values were calculated similarly using Time Weighted Average concentrations instead.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 1: Measured Concentrations in Samples taken during the Final Test and Calculated Time Weighted Average Concentrations

Loading rate (mg/L)	Measured concentration (µg/L)			TWA conc. (µg/L)
	t=0h	t=24h	t=72 h
0.010	6.33	2.6 (b)	0.16 (c)	1.8
0.032	8.91	4.70	0.16 (c)	2.7
0.10	48.7	23.4	7.94	20
0.10 (a)	57.7	46.3	4.39	27 (a)
0.32	173	146	113	140
1.0	624	478	453	490

(a) Incubated without algae; (b) estimated by extrapolation of the calibration curve; (c) actual concentration was taken as half of the LOD, which was 0.31 µg/L on the day of analysis.

Table 2: Effect Parameters based on Loading Rates (mg/L)

Parameter (mg/L)		NOELR*	NOELR^#	EL10	EL20	EL50
Growth rate	Value	<0.010	0.032	0.033	0.050	0.12
	lower 95%-cl			0.019	0.034	0.094
	upper 95%-cl			0.045	0.065	0.14

cl – confidence limit; * based on statistical significance; ^#based on biological relevance.

Table 3: Effect Parameters based on Time Weighted Average Concentrations (µg/L)

Parameter (µg/L) ^(a)		NOEC*	NOEC^#	EC10	EC20	EC50
Growth rate	Value	<1.8	2.7	3.3	6.7	25
	lower 95%-cl			1.6	3.9	19
	upper 95%-cl			5.4	9.8	35

 ^(a) Effect parameters are indicative and should be interpreted with caution as it is unknown if the analytically monitored constituent is responsible for the recorded effects; cl – confidence limit; * based on statistical significance; ^#based on biological relevance.

Table 4: Growth Rate and Percentage Inhibition for the Total Test Period

Loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Pooled controls	1.760	0.0298	12
0.010 [1.8 ]	1.722	0.0244	3	2.2^#
0.032 [2.7]	1.608	0.0317	3	8.7^#
0.10 [20]	0.973	0.3031	3	45*
0.32 [140]	0.254	0.0434	3	86*
1.0 [490]	0.071	0.1012	3	96*

[ ] – TWA concentration (µg/L), * Effect was statistically significant,^#effect was statistically significant but biologically not relevant (<10%).

 

Table 5: Growth Rate and Percentage Inhibition at Different Time Intervals 

Loading rate (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Pooled controls	12	2.339		1.746		1.194
0.010 [1.8 ]	3	2.323	0.71	1.678	3.9	1.165	2.5
0.032 [2.7]	3	2.072	11	1.513	13	1.237	-3.6
0.10 [20]	3	1.429	39	0.461	74	1.028	14
0.32 [140]	3	0.780	67	0.288	84	-0.306	126
1.0 [490]	3	0.401	83	0.028	98	-0.215	118

[ ] – TWA concentration (µg/L)

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria.

Conclusions:

The 72h-EL50 of the substance for growth rate inhibition (ErL50) was 0.12 mg/L (95% confidence interval: 0.094 - 0.14 mg/L). The 72h-ErL10 was 0.033 mg/L (95% confidence interval: 0.019 - 0.045 mg/L). The 72h-NOEL was 0.032 mg/L.

It should be noted that the CMC (used here as water solubility) reported is 310 mg/L and that it was decided to follow the WAF approach because the test item is a UVCB and was observed to be not completely soluble in test medium.
These observations in relation to the solubility appear to be contradictory and therefore as worst-case the effect data based on geometric mean measured values will be used in stead. The highest nominal WAF concentration of 1 mg/L is about a factor 310 below the measured water solubility and it is therefore assumed that the test substance was actually completely dissolved and that the measured concentration for the most abundant ion trace (m/z 420.3) is representative for the availability of the whole test item.
The 72h-ErC50 and ErC10 based time weighted average measured concentrations are 25 (19 - 35) and 3.3 (1.6 - 5.4) µg/L. These values will be used as key value for chemical safety assessment.

Executive summary:

The objective of the study was to evaluate BEROL 1872 for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOELR, EL10, EL20 and EL50 for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report 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.

The batch of BEROL 1872 tested was a light yellow liquid UVCB and was not completely soluble in test medium at the loading rates initially prepared. Water Accommodated Fractions (WAFs) were individually prepared at loading rates in the range of 0.010 to 1.0 mg/L and used as test concentrations. Test solutions were prepared by using stock solutions in acetonitrile (ACN). Since the test item is an UVCB, the solvent was completely evaporated prior to addition of the test medium to avoid modification of WAF-composition due to presence of a water-miscible solvent.

A final test was performed based on the results of a preceding combined limit/range-finding test and range-finding test. Six replicates of exponentially growing algal cultures per group were exposed to a blank and a solvent control. Three replicates per test concentration were exposed to WAFs individually prepared at loading rates of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. The initial algal cell density was 10^4 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.

Growth rate and yield inhibition increased with increasing loading rate of BEROL 1872 from 0.010 mg/L upwards, resulting in 96% growth rate and 100% yield inhibition at the highest test concentration. Statistically significant growth rate and yield inhibition was found at all test concentrations. However, inhibition of growth rate at loading rates of 0.010 and 0.032 mg/L was below 10% and therefore considered to be biologically not relevant. Samples taken from all test concentrations and the control were analysed by following a fragment ion (m/z 88.8) obtained from the most abundant ion trace (m/z 420.3). The measured concentrations increased with increasing loading rate throughout the test, which indicates proper preparation of the test solutions. In addition, the analysed fraction of the test item correlated to the recorded effects on algal growth. Since the test item is a UVCB, effect parameters were expressed in terms of loading rates and Time Weighted Average (TWA) concentrations, which are indicative for the analytically monitored fraction of the test item. The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters (based on loading rates) obtained in this study are summarized in the table below.

Parameter (mg/L)		NOELR*	NOELR^#	EL10	EL20	EL50
Growth rate	Value	<0.010	0.032	0.033	0.050	0.12
	lower 95%-cl			0.019	0.034	0.094
	upper 95%-cl			0.045	0.065	0.14
Yield	Value	<0.010	<0.010	0.013	0.019	0.040
	lower 95%-cl			0.010	0.016	0.036
	upper 95%-cl			0.016	0.022	0.045

cl – confidence limit, * based on statistical significance,^#based on biological relevance.

Table: Effect Parameters based on Time Weighted Average Concentrations (µg/L)

Parameter (µg/L)^(a)		NOEC*	NOEC^#	EC10	EC20	EC50
Growth rate	Value	<1.8	2.7	3.3	6.7	25
	lower 95%-cl			1.6	3.9	19
	upper 95%-cl			5.4	9.8	35

 ^(a)Effect parameters are indicative and should be interpreted with caution as it is unknown if the analytically monitored constituent is responsible for the recorded effects; cl – confidence limit; * based on statistical significance; ^#based on biological relevance.

It should be noted that the CMC (used here as water solubility) reported is 310 mg/L and that it was decided to follow the WAF approach because the test item is a UVCB and was observed to be not completely soluble in test medium.

These observations in relation to the solubility appear to be contradictory and therefore as worst-case the effect data based on geometric mean measured values will be used in stead. The highest nominal WAF concentration of 1 mg/L is about a factor 310 below the measured water solubility and it is therefore assumed that the test substance was actually completely dissolved and that the measured concentration for the most abundant ion trace (m/z 420.3) is representative for the availability of the whole test item.

The 72h-ErC50 and ErC10 based time weighted average measured concentrations are 25 (19 - 35) and 3.3 (1.6 - 5.4) µg/L. These values will be used as key value for chemical safety assessment.

Description of key information

The 72h-EL50 of the substance for growth rate inhibition (ErL50) was 0.12 mg/L (95% confidence interval: 0.094 - 0.14 mg/L). The 72h-ErL10 was 0.033 mg/L (95% confidence interval: 0.019 - 0.045 mg/L). The 72h-NOEL was 0.032 mg/L.

It should be noted that the CMC (used here as water solubility) reported is 310 mg/L and that it was decided to follow the WAF approach because the test item is a UVCB and was observed to be not completely soluble in test medium.

These observations in relation to the solubility appear to be contradictory and therefore as worst-case the effect data based on geometric mean measured values will be used in stead. The highest nominal WAF concentration of 1 mg/L is about a factor 310 below the measured water solubility and it is therefore assumed that the test substance was actually completely dissolved and that the measured concentration for the most abundant ion trace (m/z 420.3) is representative for the availability of the whole test item.

The 72h-ErC50 and ErC10 based time weighted average measured concentrations are 25 (19 - 35) and 3.3 (1.6 - 5.4) µg/L. These values will be used as key value for chemical safety assessment.

Key value for chemical safety assessment

EC50 for freshwater algae:

25 µg/L

EC10 or NOEC for freshwater algae:

3.3 µg/L

Additional information

The objective of the study was to evaluate BEROL 1872 for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known asPseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOELR, EL10, EL20 and EL50 for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report 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.

The batch of BEROL 1872 tested was a light yellow liquid UVCB and was not completely soluble in test medium at the loading rates initially prepared. Water Accommodated Fractions (WAFs) were individually prepared at loading rates in the range of 0.010 to 1.0 mg/L and used as test concentrations. Test solutions were prepared by using stock solutions in acetonitrile (ACN). Since the test item is an UVCB, the solvent was completely evaporated prior to addition of the test medium to avoid modification of WAF-composition due to presence of a water-miscible solvent.

A final test was performed based on the results of a preceding combined limit/range-finding test and range-finding test. Six replicates of exponentially growing algal cultures per group were exposed to a blank and a solvent control. Three replicates per test concentration were exposed to WAFs individually prepared at loading rates of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. The initial algal cell density was 10^4 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.

Growth rate and yield inhibition increased with increasing loading rate of BEROL 1872 from 0.010 mg/L upwards, resulting in 96% growth rate and 100% yield inhibition at the highest test concentration. Statistically significant growth rate and yield inhibition was found at all test concentrations. However, inhibition of growth rate at loading rates of 0.010 and 0.032 mg/L was below 10% and therefore considered to be biologically not relevant. Samples taken from all test concentrations and the control were analysed by following a fragment ion (m/z 88.8) obtained from the most abundant ion trace (m/z 420.3). The measured concentrations increased with increasing loading rate throughout the test, which indicates proper preparation of the test solutions. In addition, the analysed fraction of the test item correlated to the recorded effects on algal growth. Since the test item is a UVCB, effect parameters were expressed in terms of loading rates and Time Weighted Average (TWA) concentrations, which are indicative for the analytically monitored fraction of the test item. The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters (based on loading rates) obtained in this study are summarized in the table below.

Parameter (mg/L)		NOELR*	NOELR^#	EL10	EL20	EL50
Growth rate	Value	<0.010	0.032	0.033	0.050	0.12
	lower 95%-cl			0.019	0.034	0.094
	upper 95%-cl			0.045	0.065	0.14
Yield	Value	<0.010	<0.010	0.013	0.019	0.040
	lower 95%-cl			0.010	0.016	0.036
	upper 95%-cl			0.016	0.022	0.045

cl – confidence limit, * based on statistical significance,^#based on biological relevance.

It should be noted that the CMC (used here as water solubility) reported is 310 mg/L and that it was decided to follow the WAF approach because the test item is a UVCB and was observed to be not completely soluble in test medium.

These observations in relation to the solubility appear to be contradictory and therefore as worst-case the effect data based on geometric mean measured values will be used in stead. The highest nominal WAF concentration of 1 mg/L is about a factor 310 below the measured water solubility and it is therefore assumed that the test substance was actually completely dissolved and that the measured concentration for the most abundant ion trace (m/z 420.3) is representative for the availability of the whole test item.

The 72h-ErC50 and ErC10 based time weighted average measured concentrations are 25 (19 - 35) and 3.3 (1.6 - 5.4) µg/L. These values will be used as key value for chemical safety assessment.