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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Due to insufficient reporting of the experimental details, this study was rated with a Klimisch score of 4 (‘not assignable’). As none of the available studies can serve as a key study with a sufficiently detailed reporting justifying a Klimisch score of 1 or 2 for the given endpoint, several independent sources were used in a weight of evidence approach. ‘Using weight of evidence implies that no single study of sufficient quality and reliability exists, and that information from several independent sources is required to be able to reach a conclusion on a particular property of the substance.' (guidance document How to prepare registration and PPORD dossiers, p. 87) .
Principles of method if other than guideline:
US EPA
GLP compliance:
not specified
Test organisms (species):
Skeletonema costatum
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
13.5 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
>= 1.46 - <= 2.63 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
>= 1.46 - <= 2.63 mg/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
< 1.46 mg/L
Executive summary:

US EPA 1985 (Skeletonema costatum)


 


For 1,2,4-Trichlorbenzene, following (no) effect concentrations were reported in the Health Assessment Document for Chlorinated Benzenes for the toxicity to Skeletonema costatum:


EC50 - 24h: 13.5 mg/L


EC50 - 48h: 1.46 - 2.63 mg/L


EC50 - 72h: 1.46 - 2.63 mg/L


NOEC-96h: < 1.46 mg/L

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
other: EU Risk Assessment
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Due to insufficient reporting of the experimental details, this study was rated with a Klimisch score of 4 (‘not assignable’). As none of the available studies can serve as a key study with a sufficiently detailed reporting justifying a Klimisch score of 1 or 2 for the given endpoint, several independent sources were used in a weight of evidence approach. ‘Using weight of evidence implies that no single study of sufficient quality and reliability exists, and that information from several independent sources is required to be able to reach a conclusion on a particular property of the substance.' (guidance document How to prepare registration and PPORD dossiers, p. 87) .
Principles of method if other than guideline:
EU Risk Assessment
GLP compliance:
not specified
Remarks on result:
other: EU Risk Assessment

EU Risk Assessment


 


Depending up on test system and duration the reported EC50 values range between 1.4 mg/l (Calamari et al., 1983) and 18.9 mg/l (Broecker et al., 1984).


 


Almost all above cited studies are considered to be not valid, due to unsuitable test methods that didn't take into account the highly volatile character of 1,2,4-TCB. Thus, the real values are probably lower than most of the mentioned. It may be assumed that results from static and open system tests underestimate the toxicity due to evaporation and photolysis, respectively.


 


The only valid study was done in closed system according to an US EPA Guideline (Calamari et al., 1983): Algae EC50 (96 h): 1.4 mg/l and NOEC (96 h): 0.37 mg/l.


 


This value is in general agreement with the QSAR estimation according to the TGD (1996), which results in an algae (72-96 h) EC50 of 0.95 mg/l and the QTOXMIN (Pedersen et al., 1995) estimation, which results in an EC50 (72 h) of 0.87 mg/l. The ECOSAR model estimates the green algae EC50 (96 h) to 1.37 mg/l and the chronic value (>96 h) to 0.45 (US EPA, 1994). The QSARs predicted values fit well with the experimental data.

Executive summary:

EU Risk Assessment (2003)


 


Depending up on test system and duration the reported EC50 values range between 1.4 mg/l (Calamari et al., 1983) and 18.9 mg/l (Broecker et al., 1984).


 


Almost all above cited studies are considered to be not valid, due to unsuitable test methods that didn't take into account the highly volatile character of 1,2,4-TCB. Thus, the real values are probably lower than most of the mentioned. It may be assumed that results from static and open system tests underestimate the toxicity due to evaporation and photolysis, respectively.


 


The only valid study was done in closed system according to an US EPA Guideline (Calamari et al., 1983): Algae EC50 (96 h): 1.4 mg/l and NOEC (96 h): 0.37 mg/l.


 


This value is in general agreement with the QSAR estimation according to the TGD (1996), which results in an algae (72-96 h) EC50 of 0.95 mg/l and the QTOXMIN (Pedersen et al., 1995) estimation, which results in an EC50 (72 h) of 0.87 mg/l. The ECOSAR model estimates the green algae EC50 (96 h) to 1.37 mg/l and the chronic value (>96 h) to 0.45 (US EPA, 1994). The QSARs predicted values fit well with the experimental data.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Original reference not available.
Justification for type of information:
Due to insufficient reporting of the experimental details, this study was rated with a Klimisch score of 4 (‘not assignable’). As none of the available studies can serve as a key study with a sufficiently detailed reporting justifying a Klimisch score of 1 or 2 for the given endpoint, several independent sources were used in a weight of evidence approach. ‘Using weight of evidence implies that no single study of sufficient quality and reliability exists, and that information from several independent sources is required to be able to reach a conclusion on a particular property of the substance.' (guidance document How to prepare registration and PPORD dossiers, p. 87) .
Principles of method if other than guideline:
US EPA
GLP compliance:
not specified
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
55 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
33 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
32 mg/L
Duration:
96 h
Dose descriptor:
other: NOEL
Effect conc.:
< 8.2 mg/L
Executive summary:

US EPA 1985 (Raphidocelis subcapitata)


 


Following (no) effect concentrations were reported in the Health Assessment Document for Chlorinated Benzenes. Here, the toxicity to Raphidocelis subcapitata was assessed:


 


EC50-24h: 55 mg/L


EC50-48h: 33 mg/L


EC50-72h: 32 mg/L


NOEL-96h: <8.2 mg/L


 


 

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1995
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
GLP-Guideline study, only results are published, study report not available
Justification for type of information:
Due to insufficient reporting of the experimental details, this study was rated with a Klimisch score of 4 (‘not assignable’). As none of the available studies can serve as a key study with a sufficiently detailed reporting justifying a Klimisch score of 1 or 2 for the given endpoint, several independent sources were used in a weight of evidence approach. ‘Using weight of evidence implies that no single study of sufficient quality and reliability exists, and that information from several independent sources is required to be able to reach a conclusion on a particular property of the substance.' (guidance document How to prepare registration and PPORD dossiers, p. 87) .
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
: purity not given
GLP compliance:
yes
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Water media type:
freshwater
Total exposure duration:
72 h
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
2.2 mg/L
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
5.7 mg/L
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
5.6 mg/L
Basis for effect:
biomass
Executive summary:

Nite 2009


 


The National Institute of Technology and Evaluation (Japan) assessed the toxicitiy of 1,2,4-Trichlorbenzene to Raphidocelis subcapitata in 2009. Following (no) effect values were obtained based on growth rate:


 


EC50-72h: 5.7 mg/L


NOEC-72h: 2.2 mg/L

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not given
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Justification for type of information:
Due to insufficient reporting of the experimental details, this study was rated with a Klimisch score of 4 (‘not assignable’). As none of the available studies can serve as a key study with a sufficiently detailed reporting justifying a Klimisch score of 1 or 2 for the given endpoint, several independent sources were used in a weight of evidence approach. ‘Using weight of evidence implies that no single study of sufficient quality and reliability exists, and that information from several independent sources is required to be able to reach a conclusion on a particular property of the substance.' (guidance document How to prepare registration and PPORD dossiers, p. 87) .
Qualifier:
according to guideline
Guideline:
other: U.S. EPA, Algal Assay Procedure - Bottle Test (AAPBT)
Deviations:
yes
Remarks:
test temperature: 20 ± 1°C
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- gaschromatographic analysis of toxicant concentration after 24, 48 and 96 hours
- aqueous solutions were injected directly into a gaschromatograph

The testing flasks were closed by screw caps. The caps were pierced by stainless steel needle dipping in to the culture medium. Sampling for measurement of algal growth and toxicant concentration was made through the needle by means of a syringe.

Capped flasks were shaken for 24 hours at 20°C to let vapour and liquid phases equilibrate.

After a 24 hours equilibration period, concentrations in culture media remained almost constant. Differences in the measurements carried out at different times (at the end of the equilibration period and after 48 and 96 hours of treatment) lie in the range of acceptable analytical variability.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

Stock solution was prepared as follows: an amount of the chemical ten times higher than the saturation solubility was added to distilled water in a closed vessel, stirred for 48 hours and decanted for 24 hours. The supernatant was filtered through paper-filters and the concentration was measured.

Final solutions were made by: adding 10 ml of stock culture medium (10 times standard concentration) to different amounts of toxicant stock solution. Solutions were diluted to 100 ml with distilled water and quickly transferred into the culture flask.

Capped flasks were shaken for 24 hours at 20°C to let vapour and liquid phases equilibrate.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
The algal inoculum was added, after reaching the equilibrium concentration of the test substance (24 hours) at a starting cell concentration of 5 x 10 Exp+6 cells/l.

Algal growth was measured at 24, 48 and 96 hours by in vivo fluorescence. Results were expressed as a percentage of the growth in the control culture and graphically elaborated for the evaluation of the EC50. Only the 96 hours data are reported in the publication.
Test type:
static
Water media type:
freshwater
Total exposure duration:
96 h
Hardness:
not given
Test temperature:
in exception to AAPBT the temperature was 20 ± 1°C
pH:
not given
Dissolved oxygen:
not given
Salinity:
not given
Nominal and measured concentrations:
Initial concentrations could not be measured, due to high volatility of 1,2,4-TCB. Therefore the nominal concentrations calculated from the dilution ofthe titrated stock solutions were assumed as Ci.

Ci Ceq Ci/Ceq
1.66 0.40 4.15
3.32 0.62 5.32
6.64 1.52 4.37
9.96 2.29 4.35
13.28 2.39 3.92
16.60 2.79 5.95
23.24 4.68 4.97
4.72 (± 0.67)

Ci: initial concentration
Ceq: concentration at equilibrium calculated as a mean of the measurements after the equilibration period and during treatment
Details on test conditions:
- culture medium and test conditions were similar to the "Algal Assay Procedure-Bottle Test" (AAPBT)
=> with exception that the temperature was 20 ± 1°C


TEST SYSTEM
- Test vessel:
- Type: closed
=> by screw cap with both silicone rubber (4 mm thick) and teflon gaskets
=> The caps were pierced by stainless steel needle dipping in to the culture medium. The outer end of the needle was closed with Parafilm.

- Material, size, headspace, fill volume:
=> 2 litre spherical flasks
=> the volume of the culture was 100 ml
=> the measured medium to flask volume ratio (0.047) was low enough to avoid notable carbon dioxide deficiency

- Initial cells density: 5 x 10 EXP+6 cells/ml


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Chlorophyll measurement: Algal growth was measured at 24, 48 and 96 hours by in vivo fluorescence.


TEST CONCENTRATIONS

A
Final solutions were made by adding 10 ml of stock culture medium (10 times standard concentration) to different amounts of toxicant stock solution. Solutions were diluted to 100 ml with distilled water and quickly transferred into the culture flask.

B
Initial concentrations could not be measured, due to high volatility of 1,2,4-TCB. Therefore the nominal concentrations calculated from the dilution ofthe titrated stock solutions were assumed as Ci.

Ci Ceq Ci/Ceq
1.66 0.40 4.15
3.32 0.62 5.32
6.64 1.52 4.37
9.96 2.29 4.35
13.28 2.39 3.92
16.60 2.79 5.95
23.24 4.68 4.97
4.72 (± 0.67)
Ci: initial concentration
Ceq: concentration at equilibrium calculated as a mean of the measurements after the equilibration period and during treatment
(The algal inoculum was added, after reaching the equilibrium concentration of the test substance (24 hours) at a starting cell concentration of 5 x 10 Exp+6 cells/l.)


RANGE FINDING STUDY
- no data
Reference substance (positive control):
not required
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): no data
- Observation of abnormalities (for algal test): no data
Results with reference substance (positive control):
- reference substance not required
Reported statistics and error estimates:
no data

max tested conc. of 0% eff. mg/l: 0.37

min tested conc. of 100% eff. mg/l: 4.9

Validity criteria fulfilled:
not specified
Executive summary:

Galassi S and Vighi M (1981)


 


A modificated "Algal Assay Procedure-Bottle Test" (US EPA) for testing of volatile substances was performed. The procedure permits maintenance of a constant concentration of toxicant in the culture. The test algal Raphidocelis subcapitata was added to the test solution after reaching the equilibrium of 1,2,4-TCB between vapour and liquid phase (24 h after preparation of test solution). Both concentration of 1,2,4-TCB and algal growth rate were measured at 24, 48 and 96 hours. After 96 hours duration an EC50 of 1.4 mg/l was found. Effect concentrations at 24 and 48 hours are not stated in the publication.


 


Beside a test performed by Calamari et al. (1983) the study by Galassi and Vighi (1981) provides the only valid result on toxicity to algal according to EU Risk Assessment Report (2003). Most other studies didn't consider the volatility of 1,2,4-TCB.

Description of key information

In order to fulfill the data requirements a weight of evidence (WoE) approach (REACh Regulation (EC) No. 1907/2006, Annex XI Section 1.2) was chosen. This approach is a possible adaptation to the standard information requirements which ‘should be undertaken to maximise the use of existing data and minimise the commissioning of new in vivo testing’ (ECHA Guidance R.7a, 2017, p. 364). The WoE approach serves to reduce or avoid animal testing, which should only be performed as last resort (REACh, article 25). The individual study reports were conducted in accordance with standardized guidelines. The study reports nevertheless show some deficiencies with respect to the comprehensiveness of the presented experimental details. Therefore, a reliability of 1 or 2 and thus adequacy as key study was not assignable for any of the individual studies. Consequently, the studies were assigned a Klimisch score of 4 (according to guidance document R.4, ‘not assignable: studies or data […] which do not give sufficient experimental details […]). Collectively, these experimental studies can be used to conclude on this endpoint and to satisfy the information requirement.


 


Galassi S and Vighi M (1981)


A modificated "Algal Assay Procedure-Bottle Test" (US EPA) for testing of volatile substances was performed. The procedure permits maintenance of a constant concentration of toxicant in the culture. The test algal Raphidocelis subcapitata was added to the test solution after reaching the equilibrium of 1,2,4-TCB between vapour and liquid phase (24 h after preparation of test solution). Both concentration of 1,2,4-TCB and algal growth rate were measured at 24, 48 and 96 hours. After 96 hours duration an EC50 of 1.4 mg/l was found. Effect concentrations at 24 and 48 hours are not stated in the publication.


Beside a test performed by Calamari et al. (1983) the study by Galassi and Vighi (1981) provides the only valid result on toxicity to algal according to EU Risk Assessment Report (2003). Most other studies didn't consider the volatility of 1,2,4-TCB.


 


Nite 2009


The National Institute of Technology and Evaluation (Japan) assessed the toxicitiy of 1,2,4-Trichlorbenzene to Raphidocelis subcapitata in 2009. Following (no) effect values were obtained based on growth rate:


EC50-72h: 5.7 mg/L


NOEC-72h: 2.2 mg/L


 


US EPA 1985 (Raphidocelis subcapitata)


Following (no) effect concentrations were reported in the Health Assessment Document for Chlorinated Benzenes. Here, the toxicity to Raphidocelis subcapitata was assessed:


EC50-24h: 55 mg/L


EC50-48h: 33 mg/L


EC50-72h: 32 mg/L


NOEL-96h: <8.2 mg/L


 


US EPA 1985 (Skeletonema costatum)


For 1,2,4-Trichlorbenzene, following (no) effect concentrations were reported in the Health Assessment Document for Chlorinated Benzenes for the toxicity to Skeletonema costatum:


EC50-24h: 13.5 mg/L


EC50-48h: 1.46 - 2.63 mg/L


EC50-72h: 1.46 - 2.63 mg/L


NOEC-96h: < 1.46 mg/L


 


EU Risk Assessment (2003)


The only valid study was done in closed system according to an US EPA Guideline (Calamari et al., 1983): Algae EC50 (96 h): 1.4 mg/l and NOEC (96 h): 0.37 mg/l.


This value is in general agreement with the QSAR estimation according to the TGD (1996), which results in an algae (72-96 h) EC50 of 0.95 mg/l and the QTOXMIN (Pedersen et al., 1995) estimation, which results in an EC50 (72 h) of 0.87 mg/l. The ECOSAR model estimates the green algae EC50 (96 h) to 1.37 mg/l and the chronic value (>96 h) to 0.45 (US EPA, 1994). The QSARs predicted values fit well with the experimental data.


 


Conclusion



The EC50 and NOEC values obtained from these studies are in the same order of magnitude.


72h-EC50: 1.46 - 32 mg/L


72h-NOEC: 2.2 mg/L


96h-EC50: 1.4 mg/L


96h-NOEC: 0.37 - 1.46 mg/L


The most conservative EC50 of 1.4 mg/l (Calamari et al., 1983) and NOEC value of 0.37 mg/L (Calamari et al., 1983) are both obtained after 96h.

Key value for chemical safety assessment

EC50 for freshwater algae:
1.4 mg/L
EC10 or NOEC for freshwater algae:
0.37 mg/L

Additional information