Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
28 July 2011
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Test material form:
liquid
Details on test material:
Appearance: Straw to yellow syrup
Purity/Composition: Not indicated
Test item storage: At room temperature

Additional information
Test Facility test item number: 209099/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Specific details on test material used for the study:
[Identification: WICKENOL 535
Appearance: Straw to yellow syrup
Batch: P7728
Purity/Composition: Not indicated
Test item storage: At room temperature
Stable under storage conditions until: 17 November 2019 (retest date)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the controls

[Frequency at t=0 h, t=24 h and t=72 h
Volume 2.0 mL
Storage 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.
Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration 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.
Additionally, reserve samples of 2.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis

Test solutions

Vehicle:
no
Details on test solutions:
The batch of WICKENOL 535 tested was a straw to yellow syrup. The test item was a UVCB which was not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item.
For the combined limit/range finding test and first full test, preparation of test solutions started with individual loading rates ranging between 1.0 and 100 mg/L. A 2-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle for a period of approximately 1 day. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning through glass wool and used as test concentrations. At the end of the preparation procedure, WAFs prepared at loading rates of 22 mg/L and higher were colourless and increasingly hazy with increasing loading rates, while WAFs prepared at loading rates up to 10 mg/L were clear and colorless.
For the second full (and final) test, stock solutions were prepared in tetrahydrofuran (THF, VWR International, Belgium) at nominal concentrations ranging between 0.23 and 5.0 mg/mL. The lower stock solutions were prepared separately by proportional dilution of the highest stock solution in THF. For the preparation of each test concentration, 100 µL of a stock solution was added to 1 L of test medium resulting in test concentrations that were a factor of 10,000 lower than the stock concentrations.
After preparation, volumes of 50 mL were added to each replicate vessel of the respective test concentration containing a 1.0 mL of an algal suspension providing a cell density of 104 cells/mL.
Any residual volumes were discarded.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
0.24 mmol/L
Test temperature:
22°C
pH:
8.0-8.4
Details on test conditions:
Species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), strain: NIVA CHL 1
Source In-house laboratory culture.
Reason for selection This system is an unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.
Fresh Water Algae Culture
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 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
(NH4)6Mo7O24.4H2O 0.018 mg/L
Pre-culture 4 days before the start of the combined limit/range-finding test and the second full test and 3 days before the start of the first full test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 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
NaHCO3 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2
Reference substance (positive control):
yes
Remarks:
[State positive control substance]

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 0.009 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 0.009 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.009 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.009 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.009 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.05 mg/L
Nominal / measured:
nominal
Conc. based on:
other: WAF
Basis for effect:
other: yield
Details on results:
[C
Results with reference substance (positive control):
9.1. Combined Limit/Range-Finding Testf
9.1.1. Mean Cell Densities, Inhibition of Growth Rate and Inhibition of Yield
The mean cell densities measured during the combined limit/range-finding test are presented in Table 1. Table 2 and Table 3 present the percentages growth rate inhibition and yield inhibition per concentration, respectively.
At the end of the test, growth rate and yield were inhibited by 40 and 85%, respectively, in the highest concentration tested, i.e. a WAF prepared at loading rates of 100 mg/L. It should be noted that the replicate without algae at the highest test concentration appeared to be hazy at the end of the test.
Based on these results, samples taken from WAFs prepared at loading rates of 1.0 and 100 mg/L were analysed. The initial measured concentrations were 0.042 and 2.5 mg/L, respectively. These concentrations decreased to 0.0039 and 0.0095 mg/L, respectively, at the end of the test (see also Table 2 of the appended Analytical Report). It should be noted that the highest test concentration was seemingly oversaturated, since the initially measured concentration was 25 times higher than the estimated water solubility (see paragraph 4.5.1) and appeared to be light hazy.
All test conditions were maintained within the limits prescribed by the study plan.
Table 1
Mean Cell Densities (x104 Cells/mL) During the Combined Limit/Range-Finding Test

Time (h) WICKENOL 535
Loading rate (mg/L)
Control 1.0 10 100
0 1.0 1.0 1.0 1.0
24 2.7 n.d. n.d. 2.4
48 17.9 n.d. n.d. 10.5
72 101.3 86.4 107.2 16.0
n.d. – not determined.
Table 2
Percentage Inhibition of Growth Rate During the Combined Limit/Range-Finding Test

WICKENOL 535
Loading rate (mg/L) Mean Std. Dev. n %Inhibition
Control 1.538 0.0271 6
1.0 1.486 0.0062 3 3.4
10 1.557 0.0338 3 -1.2
100 0.921 0.0557 6 40

Table 3
Percentage Inhibition of Yield During the Combined Limit/Range-Finding Test

WICKENOL 535
Loading rate (mg/L) Mean Std. Dev. n %Inhibition
Control 100.3 8.38 6
1.0 85.4 1.61 3 15
10 106.2 10.93 3 -5.9
100 15.0 2.70 6 85
9.2. First Full Test
9.2.1. Mean Cell Densities, Inhibition of Growth Rate and Inhibition of Yield
The mean cell densities measured during the first full test are presented in Table 1. Table 2 and Table 3 present the percentages growth rate inhibition and yield inhibition per concentration, respectively.
At the end of the test, growth rate and yield were inhibited by 4.4 and 22%, respectively, in the highest concentration tested, i.e. a WAF prepared at loading rates of 100 mg/L.
Based on these results, samples taken from all test concentrations and the control were analysed. Measured concentrations were generally below the limit of quantification already at the start of the test. Overall, the measured concentrations were not following a loading rates related relationship and unrealistic increases were observed after 24 and 72 hours of exposure. Additionally, both at 24 hours and 72 hours, a response was measured in the control which was higher than most of the concentrations measured in the test samples. The cause is unknown (see also Table 3 of the appended Analytical Report). For all the above, results of the analysis for determination of exposure concentrations were considered unreliable. Due to the obvious preparation difficulties and in order to confirm the results of the first full test, it was decided to perform an additional full test and to spike the test item in test medium with a solvent to ensure a more reliable range of start concentrations.
All test conditions were maintained within the limits prescribed by the study plan.
Table 4
Mean Cell Densities (x104 Cells/mL) During the First Full Test

Time (h) WICKENOL 535
Loading rate (mg/L)
Control 2.2 4.6 10 22 46 100
0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
24 10.5 11.3 10.9 10.9 10.9 10.3 10.1
48 63.6 60.8 62.4 64.8 61.8 67.6 56.6
72 287.3 275.7 268.9 269.5 257.8 255.4 223.5

Table 5
Percentage Inhibition of Growth Rate During the First Full Test

WICKENOL 535
Loading rate (mg/L) Mean Std. Dev. n %Inhibition
Control 1.887 0.0174 6
2.2 1.873 0.0154 3 0.7
4.6 1.865 0.0116 3 1.2
10 1.865 0.0151 3 1.1
22 1.851 0.0056 3 1.9
46 1.847 0.0110 3 2.1
100 1.803 0.0222 3 4.4


Table 6
Percentage Inhibition of Yield During the First Full Test

WICKENOL 535
Loading rate (mg/L) Mean Std. Dev. n %Inhibition
Control 286.3 15.48 6
2.2 274.7 12.55 3 4.1
4.6 267.9 9.48 3 6.4
10 268.5 12.03 3 6.2
22 256.8 4.34 3 10.3
46 254.4 8.41 3 11.2
100 222.5 14.80 3 22.3
9.3. Second Full (and Final) Test
9.3.1. Measured Test Item Concentrations
The results of analysis of the samples taken during the final test are described in Table 4 of the appended Analytical Report.
Samples taken from all test concentrations and the control were analysed. The initial measured concentrations were 0.012, 0.028, 0.081, 0.18 and 0.43 mg/L, respectively, in 0.023, 0.05, 0.11, 0.23 and 0.50 mg/L. After 24 hours of exposure, no responses were observed at the three lowest test concentrations, the response observed at 0.23 mg/L was below the limit of detection and the exposure concentration at the highest test concentration was estimated to be 0.0013 mg/L. At the end of the test, exposure concentrations were all at comparable low level just above the limit of detection. This was also the case for the control, for which no reason can be given. Based on these results, the TWA concentration at the highest test concentration was calculated (see Table 7). It should be noted that, even if the exposure concentration at the highest test concentration could be extrapolated at the end of the test, it was decided to use half of the LOQ to determine the TWA, since the recorded response was lower than the response observed in both controls and nothing was measured after 24 hours of exposure. Estimated concentration was considered, consequently, unreliable.
Table 7
Measured Concentrations Versus Nominal Concentrations

WICKENOL 535
Nominal Concentration (mg/L) Measured concentration (mg/L) TWA (mg/L)
t=0h t=24h t=72 h
0.50 0.43 0.0013 0.0021 0.0089
1Half of the LOQ (i.e., 0.0040 mg/L)

9.3.2. Mean Cell Densities
Figure 1 shows growth curves at different nominal concentrations of WICKENOL 535. The individual and group mean cell densities measured at 24h intervals are given in Table 13.

Figure 1
Growth Curves at Different Concentrations of WICKENOL 535




9.3.3. Inhibition of Growth Rate and Inhibition of Yield
Table 8 shows group mean growth rates and the percentages of growth rate inhibition (total test period) whereas Table 9 shows the values at different time intervals. The group mean yields and the percentages of yield inhibition are summarized in Table 10 (see Appendix 1 for the individual values). Statistical analysis of the data is shown in Appendix 2, Appendix 3 and Appendix 4.
No statistical difference was recorded between the blank control and the solvent control. No significant differences were recorded between the values for growth rates at any of the test concentrations when compared to the pooled control groups. Inhibition of yield was statistically significant but biological not relevant (i.e., <10%) at the three highest test concentrations when compared to the pooled controls.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the highest test concentration, i.e. a TWA concentration of 0.0089 mg/L, when compared to the controls.
Table 8
Growth Rate And Percentage Inhibition For The Total Test Period

WICKENOL 535
Nominal Concentration (mg/L) Mean Std. Dev. n %Inhibition
Pooled Control 1.632 0.0190 12
0.023 1.636 0.0097 3 -0.3
0.050 1.606 0.0464 3 1.6
0.11 1.615 0.0158 3 1.0
0.23 1.623 0.0130 3 0.5
0.50 (0.0089) 1.614 0.0250 3 1.1
() – Between brackets: TWA concentration
Table 9
Growth Rate And Percentage Inhibition At Different Time Intervals
WICKENOL 535
Nominal Concentration (mg/L) n 0 – 24 h 24 – 48 h 48 – 72h
Mean %Inhibition Mean %Inhibition Mean %Inhibition
Pooled Control 12 1.551 1.630 1.714
0.023 3 1.615 -4.1 1.604 1.6 1.690 1.4
0.050 3 1.527 1.6 1.635 -0.3 1.656 3.4
0.11 3 1.570 -1.2 1.581 3.0 1.693 1.2
0.23 3 1.564 -0.8 1.536 5.8 1.769 -3.2
0.50 3 1.569 -1.1 1.571 3.6 1.704 0.6

Table 10
Yield And Percentage Inhibition For The Total Test Period

WICKENOL 535
Nominal Concentration (mg/L) Mean Std. Dev. n %Inhibition
Pooled Control 132.9 7.29 12
0.023 134.5 3.97 3 -1.2
0.050 123.5 16.60 3 7.1
0.11 126.2 5.99 3 5.1#
0.23 129.3 5.11 3 2.7#
0.50 (0.0089) 126.1 9.32 3 5.1#
() – Between brackets: TWA concentration (mg/L)
# - effect biologically not relevant (<10%)
9.3.4. Determination of Effect Concentrations
Table 11 shows the effect parameters based on the TWA concentration calculated at the highest test concentration.
Table 11
Effect Parameters

Parameter (mg/L) NOEC* NOEC** EC10 EC50
Growth rate 0.0089 0.0089 >0.0089 >0.0089
Yield 0.0501 0.0089 >0.0089 >0.0089
* - based on statistical significance, ** - based on biological relevance
1Nominal concentration (mg/L)

Reported statistics and error estimates:
1.1. Acceptability of the Test
1. In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 135).
2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35% (i.e. 6.5%).
3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (i.e. 1.2%).
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), no inhibition of growth was recorded at any of the concentrations of WICKENOL 535 tested.
The EC10 and EC50 for both growth rate inhibition (72h-ERC10 and 72h-ERC50) and for yield inhibition (72h EYC10 and 72h EYC50) were beyond the range tested, i.e. were higher that a TWA concentration of 0.0089 mg/L being considered the maximum soluble concentration of the test item in test medium.
The 72h-NOEC for growth rate inhibition was 0.0089 mg/L based on statistical significance.
The 72h-NOEC for yield inhibition was 0.0089 mg/L when based on biological relevance.
Due to the very low solubility of WICKENOL 535 in water, concentration levels that might be toxic for algae could not be reached.

Executive summary:

[The objectiveothe study was to evaluate WICKENOL 535 for its ability to generate toxic effects inRaphidocelis subcapitata(formerly known asPseudokirchneriella subcapitata)during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10and EC50for 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, 2000.

The batch of WICKENOL 535 tested was astraw to yellow syrup. The test item was a UVCBwhich was not completely soluble in test medium at the loading rates initially prepared.

For the combined limit/range finding test and first full test,Water Accommodated Fractions (WAFs) were individually prepared at loading rates ranging between 1.0 mg/L and 100 mg/L and used as test concentrations. For the second full (and final) test, a stock solution intetrahydrofuran (THF)was prepared at a concentration of 5 mg/ml. Lower stock solutionswere prepared separately by proportional dilution of the highest stock solution in THF. For the preparation of each test concentration, 100 µL of each of the stock solution was added per 1 L of test medium.

A second full (and final) test was performed based on the results of a preceding combined limit/range-finding test and a first full test. Six exponentially growing algal cultures were exposed to an untreated control and to a solvent control, whereas three replicates per group were exposed to 0.023, 0.050, 0.11, 0.23 and 0.50 mg WICKENOL 535 per litre. The initial algal cell density was 104cells/mL.The total exposure period was 72 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

No statistical difference was recorded between the blank control and the solvent control. No significant differences were recorded between the values for growth rates at any of the test concentrations when compared to the pooled control groups. Inhibition of yield was statistically significant but biological not relevant (i.e., <10%) at the three highest test concentrations when compared to the pooled controls.

Samples taken from all test concentrations and the control were analysed. The initial measured concentrations were 0.012, 0.028, 0.081, 0.18 and 0.43 mg/L, respectively, in 0.023, 0.05, 0.11, 0.23 and 0.50 mg/L. Measured concentrations decreased already within 24 hours to values below the limit of detection or to values that could only be estimated. Consequently, as no significant biological effects were observed, a TWA concentration of0.0089 mg/L was calculated for the highest test concentration.

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

The effect parameters obtained in this study are summarized in the table below.

Parameter (mg/L)

NOEC*

NOEC**

EC10

EC50

Growth rate

0.0089

0.0089

>0.0089

>0.0089

Yield

0.0501

0.0089

>0.0089

>0.0089

* - based on statistical significance, ** - based on biological relevance

1Nominal concentration (mg/L)

Due to the very low solubility of WICKENOL 535 in water, concentration levels that might be toxic for algae could not be reached.


 

paste the summary of the report]