1-Naphthol, reaction products with formaldehyde

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:

17 April 2018 to 12 June 2018

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:

2011

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples for possible analysis were taken from all test concentrations and the control at t = 0 h (2.0 mL). In addition, the filter containing the undissolved residue was kept for possible analysis.
- Sample storage conditions before analysis: Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.
- Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate concentration (WAF 22 mg t.i./L) but without algae and samples for analysis were taken at the start of the test period.
- No samples were taken after 24 and 72 hours of exposure because the concentrations measured at the start of the test were below the limit of detection.
- Preparation of test solutions started with a loading rate of 100 mg t.i./L. An overnight period of magnetic stirring was applied to ensure maximum dissolution and protonation of the test material in test medium. Afterwards the pH was adjusted from 9.5 to 8.5 using 0.1 M HCl (Merck, Darmstadt, Germany). Subsequently, the aqueous WAF was collected by filtration through a 0.45 µm membrane filter (Supor®, Pall Corporation). The filtrate was clear and of a light brown coloration and showed a pH of 8.5.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Preliminary data: The test material is designed to separate out of solution when the pH is reduced below approximately 10. The test item molecules are composed of a hydrophobic unit that, at high pH, is kept in solution by an associated anion unit of low hydrophilic strength. At lower pH this anionic unit protonates and the overall molecule solubility consequently decreases.
- Preparation of test solutions: The batch of test material was a blue liquid UVCB and not completely soluble in test medium at the loading rates initially prepared. A correction factor of 5 was used to correct for the water content of the test material, i.e. the nominal amount to be tested was multiplied with the correction factor to determine the amount of test material to be weighed for the preparation of test solution. Concentrations are expressed as “mg t.i./L” when referring to the tested fraction of the test material.

- Combined limit/range-finding test: Preparation of test solutions started with loading rates individually prepared at 1.0 to 100 mg t.i./L. A 15-minute period of magnetic stirring was applied to ensure maximum dissolution of the test material in test medium. The obtained mixtures were allowed to settle for a period of 75 minutes. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by filtration through a 0.45 µm membrane filter (RC55, Whatman). After adjusting the pH from 8.0-9.6 to 7.0-7.4 using 1 M HCl (Merck, Darmstadt, Germany), the collected WAFs were used as test concentrations. At the end of the preparation procedure, the control and the lowest test concentration were clear and colourless, while the two higher test concentrations were clear and of an increasingly dark brown colouration. After preparation, volumes of 30 mL were added to each replicate of the respective test concentration. Subsequently, 0.6 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

- Solubility tests: During the combined limit/range-finding test, strong precipitation of undissolved material was observed, which interfered with the quantification of algal cells. It was thus decided to adjust the protocol for preparing the test solutions to ensure that only a completely dissolved fraction would be tested during the final test without the formation of excessive precipitation. To this end, two tests were carried out, where the highest loading rate of test material was first magnetically stirred with test medium overnight to ensure that precipitation was as quantitative as possible. In the first test, the pH of the unfiltered solution was adjusted from 9.6 to 8.5 using 0.1 M HCl (Merck, Darmstadt, Germany) and stirred for an additional 50 minutes before filtration through a 0.45 µm membrane filter (Supor®, Pall Corporation). At the end of this test, the obtained solution was clear and slightly yellow. This was done to ensure that any unprecipitated test material could precipitate according to the lowered pH setting before the filtration step. In the second test, the filtration was done without adjustment of the pH beforehand. The obtained filtrate looked comparable to the filtrate of the first test, indicating that the adjustment of the pH did not have a major effect on the appearance or amount of precipitated test material. To ensure maximum removal of the test material fraction that would precipitate at the pH value relevant for an algal growth inhibition test, it was consequently decided to adjust the pH of the mixture before the filtration step.

- Final test: Preparation of test solutions started with loading rates individually prepared at 4.6 to 100 mg t.i./L. An overnight period of magnetic stirring was applied to ensure maximum dissolution and protonation of the test material in test medium. Afterwards the pH of the two highest test concentrations was adjusted from 8.7-9.5 to 8.3-8.5 using 0.1 M HCl (Merck, Darmstadt, Germany). The pH was chosen to be higher compared to the combined limit/range-finding test to maximise the soluble fraction of the test material in test medium. Subsequently, the aqueous WAFs were collected by filtration through a 0.45 µm membrane filter (Supor®, Pall Corporation) and used as test concentrations. At the end of the preparation procedure, the control and the two lowest test concentrations were clear and colourless, while the three higher test concentrations were clear and of an increasingly light yellow to light brownish yellow colouration. After preparation, volumes of 30 mL were added to each replicate of the respective test concentration. Subsequently, 0.6 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source: In-house laboratory culture.
- 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.
- Light intensity: 60 to 120 µE/m^2/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 Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition: NaNO3 500 mg/L, K2HPO4.3H2O 52 mg/L, MgSO4.7H2O 75 mg/L, Na2CO3.10H2O 54 mg/L, C6H8O7.H2O 6 mg/L, NH4NO3 330 mg/L, CaCl2.2H2O 35 mg/L, C6H5FeO7.xH2O 6 mg/L, H3BO3 2.9 mg/L, MnCl2.4H2O 1.81 mg/L, ZnCl2 0.11 mg/L, CuSO4.5H2O 0.08 mg/L and (NH4)6Mo7O24.4H2O 0.018 mg/L.

ACCLIMATION
- 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 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 M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition: NH4Cl 15 mg/L, MgCl2.6H2O 12 mg/L, CaCl2.2H2O 18 mg/L, MgSO4.7H2O 15 mg/L, KH2PO4 1.6 mg/L, FeCl3.6H2O 64 µg/L, Na2EDTA.2H2O 100 µg/L, H3BO3 185 µg/L, MnCl2.4H2O 415 µg/L, ZnCl2 3 µg/L, CoCl2.6H2O 1.5 µg/L, CuCl2.2H2O 0.01 µg/L, Na2MoO4.2H2O 7 µg/L and NaHCO3 50 mg/L. Hardness (Ca+Mg): 0.24 mmol/L (24 mg CaCO3/L). pH: 8.1 ± 0.2.

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:

21 - 24 °C

pH:

8.1 ± 0.2

Nominal and measured concentrations:

Nominal: WAFs prepared at loading rates of 4.6, 10, 22, 46 and 100 mg t.i./L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL tissue culture bottles
- Type: Capped
- During incubation the algal cells were kept in suspension by continuous shaking.
- Initial cells density: 1 x 10^4 cells/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- 1 extra replicate of each test group for sampling purposes after 24 hours of exposure.
- 2 replicates without algae of the WAF prepared at a loading rate of 22 mg t.i./L.

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2; according to the OECD 201 Guideline, formulated using Milli-RO water.
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test. Temperature of medium was measured continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Adjustment of pH: Yes
- Photoperiod: Continuous
- Light intensity and quality: TLD-lamps with a light intensity within the range of 119 to 121 µE.m^-2.s^-1.

EFFECT PARAMETERS MEASURED:
- Appearance of the cells were recorded the end of the final test, microscopic observations were performed on the control and all test concentrations to observe for any abnormal appearance of the algae.
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. After 24 hours of exposure in the combined limit/range-finding test, the highest test concentration had become so turbid that this disturbed spectrophotometric measurement. Therefore algal density was determined by use of a microscope and a counting chamber throughout the test for all test concentrations, except the highest test concentration which could not be quantified due to the degree of precipitation. It was decided to maintain this approach of quantifying algal cell density for the final test, in case further precipitation would be observed during the course of the test.

TEST CONCENTRATIONS
- Range finding study: The project started with a combined limit/range-finding test. Six replicates of exponentially growing algae were exposed to a control and a WAF prepared at a loading of 100 mg t.i./L. Test procedure and conditions were similar to those applied in the final test with the following exceptions: Three replicates per test group were exposed to WAFs prepared at loading rates of 1.0 and 10 mg t.i./L; one extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval; pH was only measured in the control and the highest test concentration, at the end of the test algae were not observed to verify a normal and healthy appearance; and samples for were taken from all test concentrations and the control at the start and at the end of the test.
- Results used to determine the conditions for the definitive study: Yes. After the combined limit/range-finding test, it was decided to adjust the protocol for preparing the test solutions to ensure that only a completely dissolved fraction would be tested during the final test without the formation of excessive precipitation.

DATA HANDLING
- Comparison of Average Growth Rates
The average specific growth rate for a specific period is calculated as the logarithmic increase in the biomass from the equation for each single vessel of controls and treatments:

µ i-j = (ln Xj – ln Xi) / (tj – ti) (day^-1)

Where:
µi-j = the average specific growth rate from time i to j
Xi = the biomass at time i
Xj = the biomass at time j

The average growth rate at each test material concentration is then compared with the control value and the percentage inhibition in growth rate is calculated:

%Ir = [(µC - µT) / µC] x 100

Where:
%Ir = percent inhibition in average specific growth rate
µC = mean value for average specific growth rate in the control group
µT = average specific growth rate for the treatment replicate

- Yield
The percent inhibition in yield is calculated for each treatment replicate as follows:

%Iy = [(YC – YT) / YC] x 100

Where:
%Iy = percent inhibition of yield
YC = mean value for yield in the control group
YT = value for yield for the treatment replicate

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

36 other: Loading rate (mg t.i./L)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL: 32 - 38 mg t.i./L

Key result

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

22 other: Loading rate (mg t.i./L)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on biological relevance

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

28 other: Loading rate (mg t.i./L)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % CL: 27-29 mg t.i./L

Key result

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

4.6 other: Loading rate (mg t.i./L)

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

COMBINED LIMIT/RANGE-FINDING TEST
- After 24 hours of exposure, undissolved material was observed at the highest test concentration interfering with spectrophotometric measurements to quantify algal cell densities. It was thus decided to manually quantify cell densities under a microscope. The particle formation was found to also interfere with the manual quantification of algal cells as they could not be distinguished sufficiently. As such, only the cell densities in the two lower test concentrations were quantified and are reported below. The recorded cell densities showed increasing algal growth with increasing loading rate. However, considering that cell densities could not be quantified at the highest test concentration because the algal cells were not distinguishable from the test material particles under the microscope, it is highly likely that this was also the case at the lower test concentrations and that test material particles were mistaken for algal cells.
- Samples taken from all test concentrations and the control were analysed. The initial concentrations were 0.26, 2.9 and 43 mg t.i./L in WAFs prepared at loading rates of 1.0, 10 and 100 mg t.i./L. These concentrations decreased to 1.8 to 13 % of the initial concentrations at the end of the test.
- All test conditions were maintained within the limits prescribed by the study plan.

FINAL TEST
Measured Test Material Concentrations:
- Samples taken from all test concentrations and the control were analysed. At the start of the test, no detectable test material concentrations were found in any of the samples. Consequently, the undissolved residue collected in the filter was analysed, showing that the correct test material was used for the preparation of test solutions.
- During the chemical analysis of the samples taken at the start of the final test, it was not considered that the protocol for preparing the test solutions had been changed and the dilutions as applied to the samples of the combined limit/range-finding test were used to prepare the samples for the analytical measurements. With the possibly lower concentrations in the final test, it is not unlikely that a fraction might have been quantifiable in the samples if they would not have been diluted. Consequently, an extra test was conducted, where a WAF was prepared at the highest loading rate used in the final test following the same protocol as used in the final test. A sample of this solution was analysed without dilution but no concentration was detected. Subsequently, the undissolved residue collected in the filter was analysed, showing that the correct test material was used for the preparation of the analysed solution. It was concluded that no concentrations could have been detected either in the solutions used in the final test.
- On the other hand, even though no concentrations could be measured with the analytical method, a clear dose-response was recorded in the final test indicating that a fraction of the test material remained in the WAFs tested, which was not captured by the analytical method. The analytical method used the three major peaks found in the chromatograms of the un-protonated form of the test material for analytical quantification of concentrations. The precipitated fraction was included in the analysed samples of the combined limit/range-finding test and as such was also part of the measured concentrations. In the final test, only the fraction left after complete precipitation was tested explaining why no concentrations could be measured. Considering that the test material is a UVCB, it is not possible to identify which fraction or component of the test material caused the observed inhibition of algal growth.
- Based on these results, the applied loading rates were used to determine the effect parameters.

Inhibition of Growth Rate and Inhibition of Yield
- Growth rates were in the range of the controls at loading rates up to 22 mg t.i./L during the 72-hour test period, whereas the growth rate of algae exposed to 46 and 100 mg t.i./L were increasingly reduced. Statistically significant inhibition of growth rate was found at the four highest loading rates. The effects observed at the loading rates of 10 and 22 mg t.i./L were below 10% and thus considered to be biologically not relevant.
- Inhibition of yield increased with increasing loading rate of the test material from 10 mg t.i./L upwards resulting in 99 - 100 % inhibition at the two highest loading rates at the end of the test. Statistically significant inhibition of yield was found at loading rates of 10 mg t.i./L and higher.
- Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to all loading rates when compared to the control.

Experimental Conditions:
- During the exposure period the temperature measured in the incubator was maintained at 22 °C. Temperature remained within the limits prescribed by the study plan (21-24 °C, constant within 2°C).
- Exponential growth in the control (for algal test): Yes

Results with reference substance (positive control):

- Algae were exposed for a period of 72 hours to concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L and to a control. The initial cell density was 1.0 x 10^4 cells/mL.
- Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.32 mg/L and higher.
- The EC50 for growth rate inhibition (72h-ERC50) was 1.6 mg/L with a 95 % confidence interval ranging from 1.5 to 1.6 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.
- The EC50 for yield inhibition (72h-EYC50) was 0.50 mg/L with a 95 % confidence interval ranging from 0.48 to 0.52 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L. Hence, the 72h-EYC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

For determination of the NOEL and the EL50 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 (Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm, α=0.05, one-sided, smaller) or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).
Additionally, the EL10 and EL20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
Calculation of ELx 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 loading rate of the test material.
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Table 1: Growth Rate And Percentage Inhibition For The Total Test Period

Loading rate (mg t.i./L)	Mean	Std. Dev.	n	% Inhibition
Control	1.639	0.0165	6	-
4.6	1.647	0.0039	3	-0.48
10	1.604	0.0037	3	2.1*#
22	1.588	0.0148	3	3.1*#
46	0.268	0.1701	3	84*
100	0.090	0.1561	3	95*

* Effect was statistically significant.; # Effect biologically not relevant (<10 %).

Table 2: Growth Rate And Percentage Inhibition At Different Time Intervals

Loading rate (mg t.i./L)	n	0-24 h		24-48 h		48-72 h
		Mean	% Inhibition	Mean	% Inhibition	Mean	% Inhibition
Control	6	1.434	-	1.728	-	1.754	-
4.6	3	1.552	-8.3	1.672	3.3	1.716	2.2
10	3	1.195	17	1.965	-14	1.652	5.9
22	3	1.408	1.8	1.878	-8.7	1.478	16
46	3	0.934	35	0.395	77	-0.525	130
100	3	0	100	0.261	85	0.009	99

 

Table 3: Yield And Percentage Inhibition For The Total Test Period

Loading rate (mg t.i./L)	Mean	Std. Dev.	n	% Inhibition
Control	135.6	6.63	6	-
4.6	138.8	1.64	3	-2.3
10	121.9	1.38	3	10*
22	116.3	5.15	3	14*
46	1.4	1.04	3	99*
100	0.4	0.72	3	100*

* Effect was statistically significant.

Table 4: Effect Parameters

	Parameter (mg t.i./L)	NOEL*	NOEL**	EL10	EL20	EL50
Growth Rate	Value	4.6	22	26	29	36
	Lower 95 % CL	-	-	20	23	32
	Upper 95 % CL	-	-	30	32	38
Yield	Value	4.6	4.6	21	23	28
	Lower 95 % CL	-	-	21	23	27
	Upper 95 % CL	-	-	21	24	29

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

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), the test material reduced growth rate and inhibited the yield of this fresh water algae species significantly at loading rates of 10 mg t.i./L and higher.
The EL50 for growth rate inhibition (72h-ERL50) was 36 mg t.i./L with a 95 % confidence interval ranging from 32 to 38 mg t.i./L.
The EL50 for yield inhibition (72h-EYL50) was 28 mg t.i./L with a 95 % confidence interval ranging from 27 to 29 mg t.i./L.
The 72h-NOEL for growth rate inhibition was 4.6 mg t.i./L based on statistical significance.
The 72h-NOEL for growth rate inhibition was 22 mg t.i./L based on biological relevance.
The 72h-NOEL for yield inhibition was 4.6 mg t.i./L based on statistical significance as well as based on biological relevance.

Executive summary:

The toxicity of the test material to aquatic algae was investigated in accordance with the standardised guideline OECD 201, under GLP conditions.

The test material was evaluated 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 NOEL, EL10 and EL50 for both inhibition of growth rate and inhibition of yield.

The batch of the test material tested was a blue liquid UVCB and not completely soluble in test medium at the loading rates initially prepared. A correction factor of 5 was used to correct for the water content of the test material. Concentrations are expressed as “mg t.i./L” when referring to the tested fraction of the test material. Water Accommodated Fractions (WAFs) were individually prepared at loading rates of 4.6 to 100 mg t.i./L and used as test concentrations.

A final test was performed based on the results of a preceding combined limit/range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs prepared at loading rates of 4.6, 10, 22, 46 and 100 mg t.i./L. The initial algal cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start of exposure.

A dose-related increase of inhibition of both growth rate and yield was observed at the end of the test. An inhibition of 95 and 100 % was observed at the highest loading rate tested for growth rate and yield, respectively.

Samples taken from all test concentrations and the control were analysed. At the start of the test, no detectable test material concentrations were found in any of the samples. Consequently, the undissolved residue collected in the filter was analysed, showing that the correct test material was used for the preparation of test solutions. Based on these results, the applied loading rates were used to determine the effect parameters.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Under the conditions of this study, with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), the test material reduced growth rate and inhibited the yield of this fresh water algae species significantly at loading rates of 10 mg t.i./L and higher.

The EL50 for growth rate inhibition (72h-ERL50) was 36 mg t.i./L with a 95 % confidence interval ranging from 32 to 38 mg t.i./L.

The EL50 for yield inhibition (72h-EYL50) was 28 mg t.i./L with a 95 % confidence interval ranging from 27 to 29 mg t.i./L.

The 72h-NOEL for growth rate inhibition was 4.6 mg t.i./L based on statistical significance.

The 72h-NOEL for growth rate inhibition was 22 mg t.i./L based on biological relevance.

The 72h-NOEL for yield inhibition was 4.6 mg t.i./L based on statistical significance as well as based on biological relevance.

Description of key information

Under the conditions of this study, with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), the test material reduced growth rate and inhibited the yield of this fresh water algae species significantly at loading rates of 10 mg t.i./L and higher.

The EL50 for growth rate inhibition (72h-ERL50) was 36 mg t.i./L with a 95 % confidence interval ranging from 32 to 38 mg t.i./L.

The EL50 for yield inhibition (72h-EYL50) was 28 mg t.i./L with a 95 % confidence interval ranging from 27 to 29 mg t.i./L.

The 72h-NOEL for growth rate inhibition was 4.6 mg t.i./L based on statistical significance.

The 72h-NOEL for growth rate inhibition was 22 mg t.i./L based on biological relevance.

The 72h-NOEL for yield inhibition was 4.6 mg t.i./L based on statistical significance as well as based on biological relevance.

Key value for chemical safety assessment

EC50 for freshwater algae:

36 mg/L

Additional information

The toxicity of the test material to aquatic algae was investigated in accordance with the standardised guideline OECD 201, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was evaluated 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 NOEL, EL10 and EL50 for both inhibition of growth rate and inhibition of yield.

The batch of the test material tested was a blue liquid UVCB and not completely soluble in test medium at the loading rates initially prepared. A correction factor of 5 was used to correct for the water content of the test material. Concentrations are expressed as “mg t.i./L” when referring to the tested fraction of the test material. Water Accommodated Fractions (WAFs) were individually prepared at loading rates of 4.6 to 100 mg t.i./L and used as test concentrations.

A final test was performed based on the results of a preceding combined limit/range-finding test. Six exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs prepared at loading rates of 4.6, 10, 22, 46 and 100 mg t.i./L. The initial algal cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start of exposure.

A dose-related increase of inhibition of both growth rate and yield was observed at the end of the test. An inhibition of 95 and 100 % was observed at the highest loading rate tested for growth rate and yield, respectively.

Samples taken from all test concentrations and the control were analysed. At the start of the test, no detectable test material concentrations were found in any of the samples. Consequently, the undissolved residue collected in the filter was analysed, showing that the correct test material was used for the preparation of test solutions. Based on these results, the applied loading rates were used to determine the effect parameters.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Under the conditions of this study, with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), the test material reduced growth rate and inhibited the yield of this fresh water algae species significantly at loading rates of 10 mg t.i./L and higher.

The EL50 for growth rate inhibition (72h-ERL50) was 36 mg t.i./L with a 95 % confidence interval ranging from 32 to 38 mg t.i./L.

The EL50 for yield inhibition (72h-EYL50) was 28 mg t.i./L with a 95 % confidence interval ranging from 27 to 29 mg t.i./L.

The 72h-NOEL for growth rate inhibition was 4.6 mg t.i./L based on statistical significance.

The 72h-NOEL for growth rate inhibition was 22 mg t.i./L based on biological relevance.

The 72h-NOEL for yield inhibition was 4.6 mg t.i./L based on statistical significance as well as based on biological relevance.