Lorenox™

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

17 November to 03 December 2009

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

This study was performed according to OECD Guideline 202 with GLP certificate. All validity criteria were fulfilled. The substance is adequately identified, but some data on composition is missing. This study is not reliable due to high concentration of solvent used. Indeed, a large quantity of acetone was used as solvent in this study. Because of the potential for interaction with the test chemical resulting in an altered response in the test, solvent use should be restricted to situations where no other acceptable method of test solution preparation is available. The use of solvent is not the best method at the time being. Furthermore, solvents are generally not appropriate for multiconstituent substances, like the test substance (which is a mixture of isomers), where the use of the solvent can preferentially dissolve one or more components and thereby affect the toxicity. Then, the concentration/quantity of solvent used in the treatment solutions was 5 mL/L, corresponding to 3.95 g/L (with a density of 0.79), which is 50 times higher than the recommended maximum level of solvent (below 0.1 mL/L; OECD 23) and represent almost half of the EC50 of acetone (which was reported in the ECHA disseminated dossier at 8.8 g/L). Acetone is suspected to have affected the definitive results. Some solvent/substance interactions may have occurred in this study with this high concentration of acetone.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Dates of inspection: 23-24 July 2008 / Date of signature: 19 October 2009

Specific details on test material used for the study:

- Physical state: Light yellow translucent liquid
- Storage condition of test material: Stored at room temperature protected from direct sun light

Analytical monitoring:

yes

Details on sampling:

Test item was quantified for each test item concentration, at test initiation, and at the end of test, and at the medium renewal, in the old and in the new medium (definitive test).
At each the first and the second period, duplicate set of test tubes were prepared.
The first one was used for the quantification of test item at test initiation, or in the new medium (after 24 h of testing).
The second one was kept under the same environmental conditions as the test tubes, and was used for the quantification of the test item at the end of test, or in the old medium (after 24 h of testing).

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Chemical name of vehicle: Acetone
- Method: The treatment solutions were prepared in acetone. The test concentrations were set at 120% the nominal values so that recovery of the test concentrations was more than 80% after 24 h of testing.
Range-finding test: The test concentrations were adjusted after 24 h of testing by adding 15 μL/tube of the test solutions.
Definitive test: The test solutions were renewed after 24 h of testing. The daphnids were transferred to new test tubes filled with the new test solutions, and the controls (water and solvent) were transferred similarly. The concentrations of the treatment solutions were changed so that the volume was reduced to 20 μL instead of 50 μL.
- Controls:
Water control: The control tubes made with 10 mL of reconstituted water.
Solvent control: 9.95 mL of reconstituted water and 50 μL of acetone (first period), and then 10 mL of reconstituted water and 20 μL of acetone (second period).
- Concentration of vehicle in test medium: 5 mL/L

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: Strain was received from a commercial breeder and regularly sub-cultured at the Phytosafe site. Living cells of the green algae Desmodesmus subspicatus were provided as food.
- Age: Adult females were transferred in a new volume of water on the day before the start of the test. Young daphnids produced within 24 hours were used for the tests.
- Feeding during test: Daphnids was not fed during the exposure period.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None

Hardness:

Total hardness (measured value) = 240 to 250 mg/L

Test temperature:

20.1-20.4 °C

pH:

Start of the test: 7.72-7.85
End of the test: 7.74-7.95

Dissolved oxygen:

Start of the test: 94.1%
End of the test: 93.2-96.3%

Salinity:

None

Conductivity:

No data

Nominal and measured concentrations:

Nominal concentrations: 0.16, 0.31, 0.63, 1.25 and 2.50 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 15 mL capacity glass tubes filled with 9.95 mL of reconstituted water.
- Type: The tubes were capped during the exposure period.
- Renewal rate of test solution: The test solutions were renewed after 24 h of testing. The daphnids were transferred to new test tubes filled with the new test solutions, and the controls (water and solvent) were transferred similarly.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water was freshly reconstituted from analytical grade water using stock solutions of mineral components, according to ISO 6341.
- Alkalinity: < 0.01 % NaOH
- pH (measured value): 7.6
- Na/K molar ratio (calculated value): 10/1
- Ca/Mg molar ratio (calculated value): 4/1

OTHER TEST CONDITIONS
- Photoperiod: The tubes were maintained at dark in a climatic chamber.

EFFECT PARAMETERS MEASURED:
- Immobilization: Each test tube was checked for immobilized daphnids 24 and 48 hours after the start of the test. Any abnormal behavior or appearance, if any, was reported.
- Water quality: Dissolved oxygen and pH were measured at the start and end of the test in the controls and in the highest test substance concentration, at least (definitive test only).

VEHICLE CONTROL PERFORMED: Yes

RANGE-FINDING STUDY
- One replicate unit per treatment concentration and for the control.
- Test concentrations: 0.01, 0.1, 1.0, 10.0 and 100.0 mg/L
- Results used to determine the conditions for the definitive study: The daphnids were immobilized at both 10 and 100 mg/L test item treatments. The 1.0 mg/L test item treatment gave intermediate values. No immobilization was observed at both 0.01 and 0.1 mg/L test item treatments. Based on these results, the definitive test was performed with treatment concentrations ranging between 0.16 and 2.5 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate at 0.6, 1.0 and 1.7 mg/L

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

0.71 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence intervals: 0.29 - 1.72 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.34 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence intervals: 0.16 - 0.72 mg/L

Duration:

24 h

Dose descriptor:

other: The highest tested concentration without observed effect

Effect conc.:

0.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

other: The highest tested concentration without observed effect

Effect conc.:

0.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Percentage of immobilization:
For the water and solvent controls, mean value for immobilization was 5.0%.
The percentage of immobilization was 20% as a maximum for the 0.16 mg/L test item treatment. Mean final value for the group was 10%. At 0.31 and 0.63 mg/L, the mean immobility was 60 and 85%, respectively.
All the daphnids were immobilized after 48 h of testing for both the 1.25 and 2.50 mg/L test item treatments.

Water quality:
Both the pH values and the oxygen concentrations for the test item treatments were similar to those measured in the controls. In every case the pH value of the mineral medium was between 7.5 and 8.5, and the oxygen concentration remained > 3 mg/L.

Results with reference substance (positive control):

EC50-24 h for Potassium dichromate was between 0.6 and 1.7 mg/L. Calculations gave 0.98 mg/L as the measured value. The results fulfilled the validity criteria based on Phytosafe historical data: the EC50-24 for Potassium dichromate should be between 0.6 and 1.7 mg/L.

Reported statistics and error estimates:

NOEC determinations: F values were compared to F(1,6) = 5.99 for the 5 % confidence level.
EC50 calculations:
The EC50-24 h was between 0.63 and 1.25 mg/L. Statistical analysis gave 0.71 mg/L with a 95%-confidence intervals of 0.29-1.72 mg/L.
The EC50-48 h was between 0.16 and 0.31 mg/L. Statistical analysis gave 0.34 mg/L with a 95%-confidence intervals of 0.16-0.72 mg/L.

Analytical verification of the test item treatments:

After 24 h of testing, recovery of the nominal concentrations were slightly below 80%, except for the 0.63 mg/L test item treatment.

The test tubes were tightly closed for the second period of testing so as to avoid undue loss of test material.

 

Table 6.1.3/2: Recovery of nominal treatment concentrations

Test item (mg/L)	Initial medium	Medium renewal		Final medium
		Old medium	New medium
0.16	119.3%	71.9%	118.2%	103.3%
0.31	113.1%	75.0%	125.6%	104.8%
0.63	114.0%	80.3%	119.9%	97.6%
1.25	115.6%	73.3%	100.7%	95.1%
2.50	94.3%	77.4%	#	#

#Not assessed as the test tubes were discarded after 24 h of testing

 

Table 6.1.3/3: Definitive test - Percentages of immobilized daphnids for the controls and the test item treatments

 

Groups	% immobilization after 24 h				% immobilization after 48 h
	Rep 1	Rep 2	Rep 3	Rep 4	Rep 1	Rep 2	Rep 3	Rep 4
Water control	0	0	0	0	0	20	0	0
Solvent control	20	0	0	0	20	0	0	0
Test item
0.16 mg/L	0	0	0	0	20	0	0	20
0.31 mg/L	20	20	40	20	40	60	80	60
0.63 mg/L	60	60	20	20	100	100	60	80
1.25 mg/L	40	40	80	80	100	100	100	100
2.50 mg/L	100	100	100	100	100	100	100	100

 

VALIDITY OF THE TEST RESULTS

The tests were considered as valid:

Immobilization was less than 10 % in the controls,

The dissolved oxygen concentration exceeded 3 mg/L in the control and test vessels, and was therefore greater than 6.75 mg/L on the basis of 9 mg/L as the saturation value.

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the 48-hour EC50 of the test item was calculated to be 0.34 mg/L (95% Cl: 0.16-0.72).
However, this study is not reliable due to high concentration of solvent used.

Executive summary:

A Study was performed according to OECD Guideline 202 with GLP statement, to assess the 48 h-acute toxicity of the of the test item to Daphnia magna, under static conditions.

 

Test item, prepared with acetone as solvent, was exposed to daphnids at the concentrations of 0.16, 0.31, 0.63, 1.25 and 2.50 mg/L for 48 hours. Water and solvent controls were included. There were four replicates per treatment with five daphnids per replicate, which provided a total of twenty daphnids per each treatment and control group at test initiation. The test temperature was 20.1-20.4 °C. Before definitive test, a 48-hour range-finding test was conducted and the percent immobilization was 0, 0, 0, 0, 40, 100 and 100% at 0.010, 0.10, 1, 10 and 100 mg/L, respectively.

 

After 24 h of testing, recovery of the nominal concentrations were slightly below 80%, except for the 0.63 mg/L test item treatment.

The test tubes were tightly closed for the second period of testing so as to avoid undue loss of test material.

For the water and solvent controls, mean value for immobilization was 5.0%. The percentage of immobilization was 20% as a maximum for the 0.16 mg/L test item treatment. Mean final value for the group was 10%. At 0.31 and 0.63 mg/L, the mean immobility was 60 and 85%, respectively. All the daphnids were immobilized after 48 h of testing for both the 1.25 and 2.50 mg/L test item treatments.

 

EC₅₀-24 h for Potassium dichromate was between 0.6 and 1.7 mg/L. The results fulfilled the validity criteria based on the historical data.

 

Under the test conditions, the 48-hour EC50 of the test item was calculated to be 0.34 mg/L (95% Cl: 0.16-0.72).

However, this study was not considered reliable and was disregarded due to major methodological deficiencies. Indeed, a large quantity of acetone was used as solvent in this study. Because of the potential for interaction with the test chemical resulting in an altered response in the test, solvent use should be restricted to situations where no other acceptable method of test solution preparation is available. The use of solvent is not an appropriate method of exposure to aquatic organisms except under extreme circumstances. With a solubility of approximately 44 mg/L, use of a vehicle is not necessary. Furthermore, solvents are generally not appropriate for multiconstituent substances, as is the case for this test substance (which is a mixture of isomers), where the use of the solvent can preferentially dissolve one or more components and thereby affect the toxicity. Then, the concentration/quantity of solvent used in the treatment solutions was 5 mL/L, corresponding to 3.95 g/L (with a density of 0.79), which is 50 times higher than the recommended maximum level of solvent (below 0.1 mL/L; OECD 23) and represents almost half of the EC50 of acetone (which was reported in the ECHA disseminated dossier at  8.8 g/L).Therefore, solvent/substance interactions may have occured in this study with this high concentration of acetone and the result cannot be considered fit for use.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

(Q)SAR

Adequacy of study:

key study

Study period:

August 01st, 2019 to August 13th, 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
iSafeRat® toolbox – in Silico Algorithms For Environmental Risk And Toxicity version 2.4

2. MODEL (incl. version number)
iSafeRat® holistic HA-QSAR v1.8

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
isomer 1: CC1C(OC)CCC(C)=CCCC=C(C)CC1
isomer 2: CC1C(OC)CCC(C)=CCCC(C)=CCC1
isomer 3: CC1C(OC)CCC=C(C)CCC(C)=CCC1
isomer 4: CC1CCC(C)=CCCC(C)=CCCC1OC
The toxicity of the test item was predicted using the iSafeRat® Ecotox module providing the Subcooled Liquid Water Solubility (SLWS) as the input. TThe SLWS has been predicted using the iSafeRat® Water Solubility module providing the experimental log KOW value as the input. Water Solubility of the test item = 2.09 mg/L (or -5.053 in log (mol/L).

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF

5. APPLICABILITY DOMAIN
See attached QPRF

6. ADEQUACY OF THE RESULT
See attached QPRF

Reason / purpose for cross-reference:

reference to other study

Remarks:

QSAR input value

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

QSAR model

Principles of method if other than guideline:

The purpose of this QSAR model is to accurately predict the acute toxicity to daphnids as would be expected in a laboratory experiment following OECD Guideline 202 (OECD, 2004b) and EC method C.2 (European Commission, 2008) for specific, named mechanisms of action. The model provides an in silico prediction for the 48-hour EC50 value that can effectively be used in place of an experimentally derived 48-hour EC50 value. The regression based method used to achieve this has been fully validated following the OECD recommendations (OECD, 2004a).

GLP compliance:

no

Remarks:

QSAR model

Specific details on test material used for the study:

Water Solubility: 2.09 mg/L (KREATiS, 2019)

Analytical monitoring:

no

Remarks:

QSAR model

Details on sampling:

not applicable

Vehicle:

no

Remarks:

QSAR model

Details on test solutions:

not applicable

Test organisms (species):

other: Daphnia magna, Daphnia pulex

Details on test organisms:

No difference in terms of toxic mechanism of action between invertebrate (or indeed other) aquatic species is expected. Any observed differences may be attributed to lifestyle related parameters (e.g. shell closing in molluscs) and relative duration of study versus bodysize rather than to a specific toxic mechanism causing species differences.

Test type:

other: QSAR model

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

Results from a test duration of 48 hours only were used for daphnid species.

Post exposure observation period:

None

Hardness:

The QSAR is based on data from studies performed at acceptable hardness to ensure control survival.

Test temperature:

The temperatures varied from approximately 20 to 23 °C depending on the species used to construct the algorithm. This small difference is not expected to contribute to the variability of the EC50 values found in experimental data.

pH:

Test results were taken from studies with measured pHs between 6 - 9.

Dissolved oxygen:

The QSAR is based on data from studies performed at acceptable oxygen concentrations (generally >60%).

Salinity:

Not applicable

Conductivity:

Not data

Nominal and measured concentrations:

Studies were used only where sufficient evidence was presented to determine that the substance was stable under test conditions (i.e. maintained within ± 20 % of the nominal) or, if not, the result was based on measured concentrations as geometric mean.

Details on test conditions:

Preferentially results from semi-static studies were used. However, substances tested using a static design were accepted (preferably accompanied by analytical measurements over the study period). For suspected volatile substances only tests performed in closed vessels were accepted unless accompanying analytical monitoring proved such a design was not necessary.

Reference substance (positive control):

no

Remarks:

QSAR model

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.84 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95%CI: 0.34 – 2.09 mg/L

Details on results:

The test item falls within the applicability domain of the model except for the descriptor domain. From a descriptor domain point of view, the test item falls within the intermediate domain where baseline toxicity cannot be experimentally measured accurately. According to a k-NN approach, the toxicity of the test item is estimated as the geometric mean between the toxicity value predicted using the regression line and the solubility cut-off line (with the confidence intervals being placed at these limits).

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

95% confidence interval (α = 0.05): 0.34 – 2.09 mg/L.
QSAR statistical parameters are given in the QMRF and the QPRF

Analysis of the Applicability Domain

Descriptor domain

The Subcooled Liquid Water Solubility value (-5.053 in log (mol/L)) given as the input to the Ecotox module of the iSafeRat® Holistic HA-QSAR falls within the intermediate domain of the model between a log water solubility (in log (mol/L)) of - 8.33 to - 4.70 where baseline toxicity cannot be experimentally measured accurately. In this intermediate domain, the toxicity may to be greater than the water solubility limit. For confirmation, a statistical k-NN approach (k = 3) is performed on the data of substances found to be in the intermediate domain of the model. The toxicity of the three closest neighbours based on the solubility are considered. Based on these data, either the toxicity of the test item is expected to be greater than the limit of solubility, or the toxicity is estimated by the geometric mean between the toxicity value predicted using the regression line and the solubility cut-off line. According to this analysis, the toxicity of the test item is estimated as the geometric mean between the toxicity value predicted using the regression line and the solubility cut-off line (with the confidence intervals being placed at these limits).

Structural fragment domain

All chemical groups within the molecular structure are taken into account by the model.

Mechanistic domain

Currently, the ecotoxicity module of the iSafeRat® Holistic HA-QSAR can reliably predict the aquatic toxicity for chemicals with the following mechanisms of action of toxicity (MechoA):

•          non-polar narcosis (MechoA 1.1)

•          polar narcosis of alkyl-/alkoxy-phenols (MechoA 1.2)

•          polar narcosis of aliphatic amines (MechoA 1.2)

•          cationic narcosis of quaternary ammoniums (MechoA 1.3)

•          mono-/poly-esters whose hydrolysis products are narcotics (MechoA 2.1)

•          hard electrophile reactivity (MechoA 3.1)

•          RedOx cycling of primary thiols (MechoA 4.4)

•          Proton release of carboxylic acids (MechoA 5.2)

The MechoA of molecules is predicted directly from the structure. The test item as an aliphatic ether is expected to exert a MechoA 1.1 and can be taken into account by the model.

Validity criteria fulfilled:

yes

Remarks:

partially

Conclusions:

The test item falls within the applicability domain of the model except for the descriptor domain. From a descriptor domain point of view, the test item falls within the intermediate domain where baseline toxicity cannot be experimentally measured accurately. According to a k-NN approach, the toxicity of the test item is estimated as the geometric mean between the toxicity value predicted using the regression line and the solubility cut-off line (with the confidence intervals being placed at these limits).
The 48h-EC50 of the test item to daphnids was estimated as 0.84 mg/L.
95% confidence interval (α = 0.05): 0.34 – 2.09 mg/L.

Executive summary:

A Quantitative Structure-Activity Relationship (QSAR) model was used to calculate the acute toxicity of the test item to daphnid. This QSAR model has been validated to be compliant with the OECD recommendations for QSAR modeling (OECD, 2004a) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following the Guideline for Testing of Chemicals No. 202, "Daphnia sp., Acute Immobilisation Test" (OECD, 2004b), referenced as Method C.2 of Commission Regulation No. 440/2008 (European Commission, 2008). The criterion predicted was the EC₅₀ (Median Effective Concentration), a statistically derived concentration which is expected to cause immobility in 50% of test animals within a period of 48 hours.

 

The immobility of the daphnids was determined using a validated QSAR model for the Mechanism of Action (MechoA) in question (MechoA 1.1, i.e. non-polar narcosis) (Bauer et al., 2018). The QSAR model is based on validated data for a training set of 58 chemicals derived from 48-hour test on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The test item falls within the applicability domain of the model except for the descriptor domain. From a descriptor domain point of view, the test item falls within the intermediate domain where baseline toxicity cannot be experimentally measured accurately. According to a k-NN approach, the toxicity of the test item is estimated as the geometric mean between the toxicity value predicted using the regression line and the solubility cut-off line (with the confidence intervals being placed at these limits).

The 48h-EC50 of the test item to daphnids was estimated as 0.84 mg/L.

95% confidence interval (α = 0.05): 0.34 – 2.09 mg/L.

Description of key information

iSafeRat® High-Accuracy-Quantitative Structure-Activity Relationship, KREATiS, 2019 :

48h-EC50 = 0.84 mg/L (95% confidence interval: 0.34 – 2.09 mg/L)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.84 mg/L

Additional information

One experimental study and one QSAR prediction are available to assess the short-term toxicity of the registered substance to aquatic invertebrates.

 

The experimental study (Phytosafe, 2009) was considered as not reliable and was disregarded due to major methodological deficiencies. Indeed, a large quantity of acetone was used as solvent in this study. Because of the potential for interaction with the test chemical resulting in an altered response in the test, solvent use should be restricted to situations where no other acceptable method of test solution preparation is available. The use of solvent is not an appropriate method of exposure to aquatic organisms except under extreme circumstances. With a solubility of approximately 44 mg/L, use of a vehicle is not necessary. Furthermore, solvents are generally not appropriate for multiconstituent substances, as is the case for this test substance (which is a mixture of isomers), where the use of the solvent can preferentially dissolve one or more components and thereby affect the toxicity. Then, the concentration/quantity of solvent used in the treatment solutions was 5 mL/L, corresponding to 3.95 g/L (with a density of 0.79), which is 50 times higher than the recommended maximum level of solvent (below 0.1 mL/L; OECD 23) and represents almost half of the EC50 of acetone (which was reported in the ECHA disseminated dossier at  8.8 g/L). Therefore, solvent/substance interactions may have occured in this study with this high concentration of acetone and the result cannot be considered fit for use.

The QSAR prediction (KREATiS, 2019) was considered as reliable. The QSAR model has been validated to be compliant with the OECD recommendations for QSAR modeling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following the OECD Guideline 202. The immobility of the daphnids was determined using a validated QSAR model for the Mechanism of Action (MechoA) in question (MechoA 1.1, i.e.non-polar narcosis) (Bauer et al., 2018). The QSAR model is based on validated data for a training set of 58 chemicals derived from 48-hour test on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test period. The test item falls within the applicability domain of the model except for the descriptor domain. From a descriptor domain point of view, the test item falls within the intermediate domain where baseline toxicity cannot be experimentally measured accurately. According to a k-NN approach, the toxicity of the test item is estimated as the geometric mean between the toxicity value predicted using the regression line and the solubility cut-off line (with the confidence intervals being placed at these limits). The 48h-EC50 of the test item to daphnids was estimated at 0.84 mg/L (95% CI: 0.34 - 2.09 mg/L).