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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Data waiving:
study technically not feasible
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING

Methylic Adduct is a volatile substance.

Because of the volatile nature of Methylic Adduct, conducting a meaningful Algae Growth inhibition test according to OECD TG. No. 201 was observed to be unfeasible.

The algae growth inhibition test is generally performed by the use of open vessels in order to guarantee sufficient mass transfer of CO2 from the atmosphere to the test media. Open vessels exposure system was tried with Methylic Adduct but proven to be unsuitable. In fact after 24 hours from the beginning of the exposure Methylic Adduct was no longer detected in the test media, prooving that the entire amount of the Methylic Adduct initially dosed evaporated from the water. The ErC50(24h) value obtained in that study is considered reliable and relevant in describing the actual acute toxicity of Methylic Adduct over a 24 hour-exposure, but due to the limitations related to the early disappearance of the substance from the test system, the ExC10, ExC50 and NOEC values for the 48 and 72 hour-periods are considered not correctly descriptive of the toxicity of the substance over the 48 and 72 hour-exposures.

The test was tried to be conducted in closed-system but due to the difficulties of growing algae in a sealed system several changes in the test design were made during the testing in an effort to perform a valid test: the test was tried to be conducted both without and with headspace. In both the cases an additional source of inorganic carbon (NaHCO3) was added to the water. The tests failed due to the unsufficient growth of algae in the negative control, not fullfilling the validity criteria set in the OECD TG. No. 201.
The closed exposure system was tried to be optimized by the following improvements:
- Inoculum cell density reduction;
- Increased headspace;
- CO2 Enriched headspace;
- Light reduction
but none of the above improvements (alone or in combination) supported a sufficient algae growth. The closed system-test was finally judged technically unfeasible.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
disregarded due to major methodological deficiencies
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
unsuitable test system
Remarks:
The comparison with available experimental information shows that the results of the trend-analysis understimate the actual toxicity of the test compound.
Justification for type of information:
The Category is built accoriding to the OECD Toolbox v.4.3. standard workflow with the aim to predict the toxicity to Algae of Methylic Adduct.

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
All the members of the category:
1) are surface narcotics (basing on Acute aquatic toxicity MOA by OASIS profiler);
2) discrete chemical; mono constituent (predefined); Organic (basing on Substance type profiler)
3) have no alert in regard of protein binding (Basing on Protein binding by OASIS profiler)
4) own one or more of the following chemical elements in their molecule:
Carbon C;
Oxygen O;
Halogens Cl;
Halogens F;
Halogens F,Cl,Br,I,At.

According to OECD Toolbox v.4.3. standard workflow, the algae acute toxicity of the members of the category follows a trend based on Log Kow values.
The algae acute toxicity of Methilic Adduct can therefore be predicted basing on the following equation:
EC50, IC50, LC50 = 2,14 (±1,05) + 0,811 (±0,358) * log Kow, log(1/mol/L)

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL )
See attached documents:
* Prediction reprot
* Data matrix
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Principles of method if other than guideline:
Prediction in OECD QSAR toolbox V4.3.1 using trend analysis
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
4.654 other: log(1/mol/L)
Basis for effect:
other: Growth
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
5.9 mg/L
Basis for effect:
other: Growth
Details on results:
The category was built according to the OECD Toolbox v.4.3. standard workflow.
The primary categorization was based on the Acute aquatic toxicity MOA by OASIS profiler: Methylic Adduct and all the category members are basesurface narcotics. The initial prediction provided not acceptable statistics (R^2 = 0.369).
Therefore, further refinements was applied in order to improve statistics, by the application of the automatically identified subgrouping criteria. Subgrouping was applied basing on the following criteria, providing a final R^2 = 0.649:
* Substance type (subcategorization): Discrete chemical; Mono constituent (predefined); Organic
* Protein binding by OASIS (subcategorization): no alert found
* Chemical elements (subcategorization): Group 14 - Carbon C; Group 16 - Oxygen O; Group 17 - Halogens Cl; Group 17 - Halogens F; Group 17 - Halogens F,Cl,Br,I,At

The applied trend analysis provided the ErC50(72-96h) of Methylic Adducut is 5.9 mg/L (Predicted value = 4,60 log(1/mol/L), conf.range: (3,24 ; 5,96) at 95,0%).
The prediction is based on a calculated logKow of 3.04.
Conclusions:
The Algae ErC50(72-96h) of Methylic Adduct is expected to be in the range 1-10 mg/L.
Executive summary:

The algae toxicity of Methylic Adduct was estimated by the use of OECD Toolbox v.4.3 trend analysis.

The category was built according to the OECD Toolbox v.4.3. standard workflow.

The primary categorization was based on the Acute aquatic toxicity MOA by OASIS profiler: Methylic Adduct and all the category members are basesurface narcotics. The initial prediction provided not acceptable statistics (R^2 = 0.369).

Therefore, further refinements was applied in order to improve statistics, by the application of the automatically identified subgrouping parameters. Subgrouping was applied basing on the following criteria, providing a final R^2 = 0.649:

* Substance type (subcategorization): Discrete chemical; Mono constituent (predefined); Organic

* Protein binding by OASIS (subcategorization): no alert found

* Chemical elements (subcategorization): Group 14 - Carbon C; Group 16 - Oxygen O; Group 17 - Halogens Cl; Group 17 - Halogens F; Group 17 - Halogens F,Cl,Br,I,At

The applied trend analysis provided the ErC50(72-96h) of Methylic Adducut is 5.9 mg/L (Predicted value = 4,60 log(1/mol/L), conf.range: (3,24 ; 5,96) at 95,0%).

The prediction is based on a calculated logKow of 3.04.

In addition, other predictions were tried, in order to check if a different selection of profilers would have provided better statistics. The best achievable statistic was comparabe with the one of the reported prediction and all the estimations provided values in the range 1 to 10 mg/L.

Basing on the applied OECD Toolbox v.4.3 trend analysis

the Algae ErC50(72 -96h) of Methylic Adduct is expected to be in the range 1-10 mg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
August 2018 to January 2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study was conducted according to national standard method and GLP, however, due to the difficult nature of the test item it was not possible to mantain a stable concentration during exposure and at 24h the test item was no longer present in the test system. The results are however considered suitable for evaluating the Algae acute toxicity of the test compound.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Adopted 23 March 2006, Annex 5 corrected 28 July 2011
Deviations:
yes
Remarks:
Due to the difficult nature of the substance, although the duration of the test was 72h, algae were actually exposed for period < 72 hours.
Qualifier:
according to guideline
Guideline:
other: OECD Series on Testing and Assessment, No. 23: Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals
Version / remarks:
2019
Deviations:
not applicable
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: D653 22_02_2018 (manufactured by the Sponsor)
- Expiration date of the lot/batch: Feburary 2023
- Purity test date: 2018/02/27
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All the tested concentrations (1.0, 3.2, 10, 32 and 100% Saturated solution) and the control were analytically verified.
For fresh test media samples (0 hours) additional analytical vessels were prepared and sampled. This measure was set for the closed system test design and mantained in the final test conducted in standard open conditions. The reason was the volatile nature of the compound, in this way it would have been avoided to sample test vessels reducing the media volume and creating a head space for the test substance to volatilise into.
At the start of the test (0 hours), samples were taken from the control and each test concentration for chemical analysis. Analytical vessels for 0 hour analysis only were prepared in the same manner as the test vessels, however, the vessels were not inoculated with algae given that 0 hour samples are usually taken from test media preparation flasks prior to the addition of algae.
At 24 hours, samples for chemical analysis were taken from the old test media from the control and each test concentration. Two replicate test vessels were sampled and analysed individually for the control and each test concentration.
Analytical samples were not taken at 48 or 72 hours as no test substance was present in the 24 hour analytical samples.
In each case duplicate samples were taken, one for chemical analysis and one as a ‘back-up’ should further analysis be required.
- Analytical sampling method: 1 mL samples were taken directly into 9 mL of methanol and rìtransferred to analytical department.
- Sample storage conditions before chemical analysis: not applicable, the samples were directly analysed.

- Biological sampling method: At approximately 24-hour intervals after the start of the incubation period, pre determined volumes of test media (1.0 mL at 24 hours and 0.5 mL at 48 and 72 hours) were removed from each incubated test vessel, and transferred to individually identified cell counting vials. The contents of each vial were diluted to a 10 mL final volume with an electrolyte solution. The cell density of the vial contents was then determined using a particle counter).
Vehicle:
no
Details on test solutions:
Due to the difficoult nature of the test substance, extensive work was conducted in order to define the best procedure for media preparation. (The work is described in the study report No. 3202248 "Methylic Adduct: Acute Toxicity to Daphnia magna", IUCLID section 6.1.3.).
The scope of the preliminaty work was to find a procedure for solubilizing the test item reducing as much as possible losses by evaporation.
The procedure described below is based on the results of the media preparation trials conducted as part of the study No. 3202248 "Methylic Adduct: Acute Toxicity to Daphnia magna".

At the start of the test, an amount of test substance (35 mL) was added to 5500 mL of EC medium in a completely filled and sealed Duran bottle fitted with a specialist lid allowing removal of media from the mid-point without exposing media to the atmosphere. The preparation was then stirred for ~24 hours followed by a settle period of ~24 hours. Saturate was then removed from the midpoint of the vessels using a gas tight syringe. The final media were prepared by adding sufficient EC media to each individual vessel and then filling up with saturate to achieve the required concentration. A control treatment was prepared by adding EC medium only to the control vessels.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Oban, UK
Semi-axenic cultures of Pseudokirchneriella subcapitata were maintained in liquid culture. These were used to inoculate starter liquid cultures, which in turn were used to inoculate test vessels containing control and test media at the start of each test.
The axenic strain of Pseudokirchneriella subcapitata (CCAP 278/4) was obtained from a concentrated liquid slope culture from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Oban, UK. On receipt from the CCAP, the slope culture was stored in the fridge at 2 8°C. A typical shelf life for each slope was 8 months, after which the slopes were discarded.
----------

Prior to testing, duplicate starter cultures were prepared and incubated under test conditions (as detailed below) to obtain sufficient algal cells in exponential growth and to achieve a starting algae cell density of 1 × 104 cells/mL.

Inoculation of Test Cultures
Prior to each test, a sub-sample (ca 200 L) of a current liquid slope culture was added to two starter cultures (conical flasks) containing 100 mL EC medium.
Each culture was incubated (under the same conditions as used in the test) for at least 72 hours prior to the start of each test. Following incubation, the cell density of a single starter culture was established using a haemocytometer and microscope.
An inoculum volume was then calculated and added to each test vessel, to achieve a starting alga cell density of 1 × 104 cells/mL. All flasks for a particular test were inoculated from a single starter culture.
Regular tests are conducted using a reference toxicant to ensure that cultures are of the highest quality and sensitivity. The results from the latest reference test are presented in the Annex.


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
Due to the high volatility of the test item it was not possible to mantain a stable concentration during the 72h exposure. Cell density was measured over the 72h period but at 24h no more test item was present in the test media.
Post exposure observation period:
not applicable
Test temperature:
The incubator temperature was set within the range 21 to 24°C and maintained within ± 2°C for the duration of the test. The temperature was recorded continuously using a digital thermometer.
The temperature range during the test was 22.4 to 23.1°C.
pH:
The pH of the test solutions ranged from 7.44 to 7.80 at test initiation in solutions with algae. The pH tended to increase relative to increases in algal densities, which is typical for tests conducted with P. subcapitata. The pH in the control increased by a maximum of 0.18 units.
Nominal and measured concentrations:
Nominal: 1.0, 3.2, 10, 32, 100 % saturated solutions.
Measured conc. at 0 hours: Measured conc. at 24 hours:
Geometric mean of measured conc.: 0.0316 mg/L (1%SS), 0.0730 (3.2%SS), 0.157 (10%SS), 0.257 (32%SS), 0.448 mg/L (100%SS).
The geometric mean measured concentrations were calculated using 0-hour measured concentrations (or a value of half LOQ where the measured concentration was Ref: ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7b: Endpoint specific guidance Version 4.0 June 2017
Details on test conditions:
Due to the volatile nature of the compound the test was initially tried to be conducted in a sealed system, as per guidance from OECD 23. Due to the difficulties of growing algae in a sealed system several changes were made in an ongoing manner during the testing in an effort to perform a valid test. Initially testing was conducted in a sealed system, however, after a failed attempt a head space was then added to allow for better movement of the algae within the vessel and allow a certain degree of gaseous exchange. A headspace was created by removing 1, 2 and 5 mL from each test and control vessel over several tests. In addition, modifications to the test design such as increased carbon content of the media, reduced inoculum concentration and reduced light intensity were also investigated. However, these modifications did little to improve the validity of the tests. Therefore, the decision was made to run the test in an open system.
Initially test vessels were dosed using direct addition of test substance to each individual vessel, however, this resulted in test substance being present at the base of the vessels, suggesting test substance solubility issues. The sponsor later confirmed the most appropriate way to prepare the test substance was using a sealed saturated solution preparation.
The details of the first ten failed definitive tests are summarised in the attached table.

The details of the succesfully definitive test are summarised below:
TEST SYSTEM
- Test vessel: 250 mL conical flasks with foam bung lids
- Type: open
- Material: glass
- Fill volume: 100 mL
- Initial cells density: 1 × 10E4 algae cells/mL
- Control end cells density: 35779 algae cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
Standard algal (EC) culture medium.
To prepare the algal culture medium, stock solutions containing the various nutrients were prepared with reverse osmosis (RO) water. Aliquots of each of the stock solutions were then added to RO water. The algal culture medium was autoclaved.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 24/24h
- Light intensity and quality: constant light (4440 to 8880 Lux) generated by using LED lights, emitting light across the visible portion of the spectrum (400 - 700 nm). The light intensity within the test area was monitored at the start and end of the test over five areas within the test area.
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [electronic particle counter]
A single sample taken from each of the incubated test vessels and diluted with an appropriate counting medium. Each sample counted twice, and a mean sample cell count determined. Counts performed at 24, 48 and 72 hours.
Observation of test media appareance.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2

- Range finding study
- Test concentrations: 1, 10 and 100 mg/L (nominal), corresponding to 1.28 , 0.722 and 0.887 mg/L (measured).
- Results used to determine the conditions for the definitive study: Partially.
The chemical analysis during the range-finding test suggested that the test substance was not stable in the test system and that there were potential solubility related issues. The media preparation procedure was consequently changed. The results of the range-finding test suggested that the 72-hour EC50 values for yield and specific growth rate were likely to be >100 mg/L, based on nominal test substance concentrations but basing on the solubility and chemical issues observed during the range-finding test the results were not considered fully reliable.
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
< 0.032 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
0.018 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
24 h
Dose descriptor:
EC20
Effect conc.:
0.084 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
0.214 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.073 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
0.092 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
48 h
Dose descriptor:
EC20
Effect conc.:
0.157 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.209 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.73 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.117 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.165 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.224 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Despite the analytical results indicating that the test substance was either no longer in the test system or at levels below the limit of detection, the 72-hour effect results are considered to be due to the early exposure of the test organism to the test substance as no growth recovery was observed in the affected concentrations after 72 hours.

Full results are reported in the attached background material.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
Confidence limits associated to the calculated effect levels are reported in the attached background material.

The observed effect on Algae growth is considered to due to the early exposure of Algae to the test compound. The comparison of the reduction in average specific growth rates in the three time periods 0 – 24 hours, 0 - 48 hours and 0 - 72 hours (table below)shows in fact that the growth inhibition occurred during the first 24 hours of exposure when the substance was still present in the system and that simply the Algae growth did not recover once that the test substance was no longer present in the media. The most relevant effect level representing the actual acute toxicity of the test compound is therefore considered to be the ErC50 at 24 hours.

Due to the fact that the Algae test is a multigeneration study, it is generally accepted that a 72-hour (or longer) EC10 or NOEC value can be considered as a long-term result. In the present study, however, the reported ErC10(72h) and NOEC(72h) are considered to undestimate the actual chronic toxicity of the substance as they are based on the geometric mean of measured concentrations calculated on a 72 hour-exposure period (geometric mean of 0 -hour measured concentrations (or a value of half LOQ where the measured concentration was <LOQ) and the LOD) where in the reality the substance was already not detected at 24 hours.

The use of the ErC10(24h) and NOEC(24h) as long term results is considered not appropriate as the duration of the period is not long enough for considering the test a multigeneration chronic toxicity study.

Nominal Concentration

(% saturated solution)

Reduction in average specific growth
 rates relative to the control (%)*

 

0-24 hours

0-48 hours

0-72 hours

 

Control

NA

NA

NA

 

1.0

17.594

0.296

3.222

 

3.2

18.908

6.924

3.705

 

10

27.079

20.076

15.586

 

32

66.352

76.631

66.110

 

100

90.146

82.711

89.106

 

NA

Not applicable

Validity criteria fulfilled:
yes
Conclusions:
Due to the early disappearance of the substance from the test system the most relevant effect level representing the actual acute toxicity of the test compound is considered to be the ErC50 at 24 hours, equal to 0.214 mg/L. No reliable chronic effect levels were derived under the present study.
Executive summary:

The test was conducted in accordance with OECD Chemicals Testing Guideline No. 201. Alga, Growth Inhibition Test (adopted 23 March 2006, Annex 5 corrected 28 July 2011) and OECD Guidance Document (OECD Series on Testing and Assessment, No. 23 (2019); Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals).

The test was conducted at the nominal test concentrations of 1.0, 3.2, 10, 32 and 100 % saturated solution.

Due to the volatile nature of the compound the test was initially tried to be conducted in sealed system, but due to the difficulties of growing algae in a sealed system several changes in the test design were made in an ongoing manner during the testing in an effort to perform a valid test. The test was finally conducted in standard open conditions due to the impossibility of having a valid test in closed system.

As expected, chemical analysis conducted during the test showed that no more test substance was present the media at 24 hours, therefore the results were based on both nominal exposure concentrations and geometric mean measured concentrations. The geometric mean measured concentrations were calculated using 0-hour measured concentrations (or a value of half LOQ where the measured concentration was <LOQ) and the LOD.

Despite the substance was no more detectable in the test media after 24hours from the beginning of exposure, a concentration-related effect on algae growth was still present at the conclusion of the test at 72 hours from the beginnig. The reduction in average specific growth rates in the three time periods 0 – 24 hours, 0 - 48 hours and 0 - 72 hours show that the growth inhibition occurred at the beginning of the exposure period when the test substance was still present in solution and the algae did not recover once the test substance was no longer present in the system. The most relevant effect level representing the actual acute toxicity of the test compound is therefore considered to be the ErC50 at 24 hours, equal to 0.214 mg/L.

Due to the limitations related to the early disappearance of the substance from the test system, no reliable chronic effect levels were derived under the present study.

Description of key information

The ErC50(24h) of Methylic Adduct was esperimentally determined to be 0.214 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.214 mg/L

Additional information

Methylic Adduct is a volatile substance.

Because of the volatile nature of Methylic Adduct, conducting a meaningful Algae Growth inhibition test according to OECD TG. No. 201 was observed to be unfeasible. In fact, due to the volatile nature of the Methylic Adduct, the Algae growth inhibition test was initially tried to be conducted in sealed system, but the test was technically unfeasible because of the impossibility of achieving a valid test in a such conditions. The study was therefore tried to be conducted in standard (open) conditions. The study fullfilled all the validity criteria and it is therefore considered valid but it only provided partially meaningully information as after 24 hours from the beginning of the exposure Methylic Adduct was no longer detected in the test media prooving that the entire amount of the Methylic Adduct initially dosed evaporated from the water.

The ErC50(24h) value obtained in that study is considered reliable and relevant in describing the actual acute toxicity of Methylic Adduct over a 24 hour-exposure, but due to the limitations related to the early disappearance of the substance from the test system, the ExC10, ExC50 and NOEC values for the 48 and 72 hour-periods are considered not correctly descriptive of the toxicity of the substance over 48 and 72 hour-exposures.

In addition to the above, the EC50 (72-96h) value for the algae growth inhibition was estimated by the use of OECD toolbox v.4.3 trend analysis. Several runs based on different criteria for categorization and grouping were carried out . All the estimations provided values in the range 1 to 10 mg/L showing that the applied analysis undertimates the actual toxicity of the substance.