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Toxicity to aquatic plants other than algae

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Endpoint:
toxicity to aquatic plants other than algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
no guideline followed
Principles of method if other than guideline:
Vegetative shoots of Elodea canadensis were allowed to grow at four different test concentrations in a replicated aquatic community (model ecosystem) for 28 d. Growth and productivity of Elodea were recorded and NOEC was calculated.
GLP compliance:
no
Remarks:
the study was conducted prior to GLP.
Analytical monitoring:
yes
Details on sampling:
Please see the section below. The study report does not include details about sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION: Preparation of test solutions is not reported. The test concentrations were delivered to the duplicate test vessels by a modified Mount and Brungs 1 L proportional diluter.
Test organisms (species):
other: Elodea canadensis
Details on test organisms:
TEST ORGANISM
- Common name: Elodea
- Strain: Not reported
- Source (laboratory, culture collection): Not reported
- Age of inoculum (at test initiation): vegetative shoots (10 cm long)
- Method of cultivation: Not reported

ACCLIMATION
- Acclimation period: 3 d
- Culturing media and conditions (same as test or not): Not reported
- Any deformed or abnormal cells observed: Not reported
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
28 d
Post exposure observation period:
None
Hardness:
120 mg/L CaCO3
Test temperature:
21 ± 2°C
pH:
Mean pH: 8.1 ± 0.2
Dissolved oxygen:
Mean Dissolved Oxygen Concentration (DOC): 7.8 mg/L (ranged, 7.0-9.0)
Nominal and measured concentrations:
Nominal test concentrations were: 0, 0.5, 1.0, 2.0, and 4.0 mg/L. MBAS confirmed the expected nominal concentrations. Two figures in the study report show the confirmation of nominal test concentrations, but details not provided.
Details on test conditions:
TEST SYSTEM
- Incubation chamber used: Not reported
- Test vessel: Aquaria containing 2.5 cm of natural lake sediment
- Type: Open
- Material: 19 L glass
- Type of cover: Not reported
- Aeration: No (test was flow-through)
- Agitation: No
- Type of flow-through: Proportional diluter
- Renewal rate of test solution: 8 replacement volumes/d
- Control end cells density: Not reported
- No. of shoots per vessel: 8 vegetative shoots
- No. of frond per colony: Not reported
- No. of vessels per concentration (replicates): Two
- No. of vessels per negative control (replicates): Two :
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: No
- Detailed composition if non-standard medium was used: Not reported

SEDIMENT USED (for rooted aquatic vascular plants)
- Origin: Winton Lake, Cincinnati, Ohio
- Textural classification (% sand, %silt and clay): Not reported
- organic carbon (%): Not reported
- Geographic location: Cincinnati, Ohio

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was carbon and reverse-osmosis filtered well water
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals (mg/L): Zinc: <0.001; Lead: <0.01; Iron: <0.05; Copper: <0.001,
- Nitrate (mg/L): <0.05
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: Not reported
- Culture medium different from test medium: No
- Intervals of water quality measurement: Twice weekly
- Other: Temperature was monitored continuously with a recording thermograph

OTHER TEST CONDITIONS
- Sterile test conditions: Not reported
- Adjustment of pH: Not reported
- Photoperiod: Not reported
- Light intensity and quality: Not reported

EFFECT PARAMETERS MEASURED: Includes growth (as total length) and productivity (as ash-free dry weight) of the Elodea at the end of 28 d exposure period.
- Determination of frond number: Not reported
- Determination of biomass: Yes, dry weight
- Determination of frond area: Not reported

NEGATIVE CONTROL PERFORMED: Yes (received only filtered well water)

SAMPLING
- Sampling frequency: Only on Day 28, as sampling of test species was not done during exposure period.

RANGE-FINDING STUDY: No
Reference substance (positive control):
no
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 4 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: Nominal concentration confirmed by analytical measurement.
Details on results:
- Any visual signs of phytotoxicity (abnormalities): Not reported
- Decrease in frond size: Not reported
- Necrosis / chlorosis: Not reported
- Sinking of fronds: Not reported
- Other: The growth and productivity was not inhibited at any test concentration, including the highest (4.0 mg/L).
- Any stimulation of growth found in any treatment: Growth throughout the exposure period approximately doubled the initial biomass of the vegetative shoots used at the start of the exposure (Initial biomass = 37 mg ashed-free dry weight; overall mean for all treatment concentrations = approx. 72 mg ashed-free dry weight). Overlap of 95% confidence limits for all treatments with unexposed control production confirms the lack of LAS effects throughout the exposure period.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not reported
- Effect concentrations exceeding solubility of substance in test medium: No
- Bioconcentration factor: Mean bioconcentration factor 629.5 based on a wet weight basis
Results with reference substance (positive control):
Positive control was not included in the study.
Reported statistics and error estimates:
Not reported
Validity criteria fulfilled:
not applicable
Conclusions:
The 28 d NOEC of C11.6 LAS to Elodea canadensis in a model ecosystem study was > 4 mg/L.
Executive summary:

The long term toxicity of C11.6 LAS (linear alkylbenzene sulfonate) to the aquatic plant (Elodea canadensis) was evaluated in a 28 day model ecosystem flow-through test. The nominal test concentrations were 0, 0.5, 1.0, 2.0, and 4.0 mg/L, confirmed by analytical measurements.

The growth and productivity of the Elodea was recorded after the exposure period of 28 d.

Growth inhibition was not observed even at highest tested concentration (4 mg/L). Growth throughout the exposure period approximately doubled the initial biomass of the vegetative shoots used at the start of the exposure.  The initial biomass was 37 mg ashed-free dry weight; the overall mean for all treatment concentrations was 72 mg ashed-free dry weight. Overlap of 95% confidence limits for all treatments with unexposed control production confirmed the lack of LAS effects throughout the exposure period.

Hence, the NOEC was found to be >4 mg/L.

Endpoint:
toxicity to aquatic plants other than algae
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From July, 1978 to November, 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Remarks:
pre-dates GLPs
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Analytical monitoring:
yes
Details on sampling:
Not reported
Vehicle:
no
Details on test solutions:
Not reported
Test organisms (species):
Lemna minor
Details on test organisms:
TEST ORGANISM
- Common name: Duckweed
- Strain: Not reported
- Source: The test species was originally collected near Boca Raton, Florida, and has been in continuous culture at the EPA Environmental Research Laboratory, Duluth, Minn., since 1971.
- Age of inoculum (at test initiation): Not reported
- Method of cultivation: Test species stocks were maintained in X0.01 Hutner's solution in a flow through system

ACCLIMATION
- Acclimation period: Not reported
- Culturing media and conditions (same as test or not): Not reported
- Any deformed or abnormal cells observed: Not reported
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Post exposure observation period:
None
Hardness:
120-130 (as CaCO3 ) mg/L
Test temperature:
21-23 °C
pH:
7.2-7.6
Dissolved oxygen:
8.5 mg/L
Nominal and measured concentrations:
Nominal concentration: Not specified in the study report. All results are based on measured active ingredient. Details of analytical results are not provided in the study report. The test concentrations, based on measured active components, were: 0, 2.1, 3.8, 8, 17 and 34 mg/L.
Details on test conditions:
TEST SYSTEM
- Incubation chamber used: Incubators were not used. However, the test chambers were placed in environmental control housing with a removable front cover for servicing.
- Test vessel: Pyrex glass test chambers
- Type: Open
- Material: All glass test chambers were separated into four individual compartments, each measuring 1.5 x 10.8 x 6.8 cm. Water volume in each compartment was regulated to approx. 400 mL
- Type of cover: Not reported
- Aeration: No (flow-through test)
- Agitation: No
- Type of flow-through: Proportional diluter
- Renewal rate of test solution: Water flow into the diluter was 1.5 L every 16 minutes, which was mixed with 6 mL nutrient stock to give the final concentration of 0.01X Hutner's solution in the test chambers. The flow rate through the individual test chambers was approximately 14 replacement volumes/day.
- Control end cells density: Not reported
- No. of colonies per vessel: Seven (two-frond, root-excised duckweed colonies)
- No. of fronds per colony: Two
- No. of vessels per concentration (replicates): Four
- No. of vessels per negative control (replicates): Four

GROWTH MEDIUM
- Standard medium used: No
- Detailed composition if non-standard medium was used: Nutrient stock solution used was X2.5 Hutner's solution which was mixed with dilution water to give the final concentration of X0.01 in the test chambers. Detailed composition of growth medium is not reported in the study.

SEDIMENT USED: Sediment was not used as the test species is a floating plant

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was carbon- and reverse osmosis filtered well water, with added nutrients.
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals (mg/L): Mercury = <0.005, Cadmium = <0.005, Zinc = <0.001, Lead = <0.005, Copper = <0.001, Iron = <0.05, Sodium = 11.6, Nickel = <0.033
- Pesticides (mg/L): Chlorinated insecticides= <0.00005, Organophosphate insecticides = <0.0005
- Nitrite: <0.05 mg/L
- Nitrate: <0.05 mg/L
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: Not reported
- Culture medium different from test medium: No
- Intervals of water quality measurement: Not reported

OTHER TEST CONDITIONS
- Sterile test conditions: Not reported
- Adjustment of pH: Not reported
- Photoperiod: Continuous illumination
- Light intensity and quality: 3875 lux (360 foot candles) fluorescent lights

EFFECT PARAMETERS MEASURED
- Determination of frond number: Number of fronds in each replicate chamber was recorded once every 24 h for 7 d. Every frond visibly projecting beyond the edge of the parent frond was counted manually.
- Determination of biomass: Dry weights were determined at the end of exposure period.
- Determination of frond area: Not determined
- Other: The root lengths were determined at the end of the exposure period. Where possible, 15 to 20 roots from each replicate weremeasured to the nearest 0.5 cm and the ten largest values were selected for statistical analysis. All plant material from each replicate was placed in a tared aluminum dish and dried at 103°C for 3 h. Since the growth rates stabilized by the Day 4, only frond count data from Days 4 through 7, and Day 7 root length and dry weight data were used for statistical analysis. From the frond count data, two other parameters growth rate and doubling time were computed or Day 4 through 7.

NEGATIVE CONTROL PERFORMED: Yes (received only carbon and reverse osmosis filtered well water)

RANGE-FINDING STUDY: No
Reference substance (positive control):
no
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
0.24 mg/L
Basis for effect:
frond number
Remarks on result:
other: EC10 was normalized to reflect the toxicity of LAS with a carbon chain length 11.6
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
3.6 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 2.9-4.3
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
4.9 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
other: root length
Remarks on result:
other: 0-12.6
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
4.8 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks:
(Doubling time)
Remarks on result:
other: 4.4-5.2
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
0.21 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
frond number
Remarks on result:
other: 0.16-0.38
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
0.61 mg/L
Basis for effect:
frond number
Remarks on result:
other: EC20 was normalized to reflect the toxicity of LAS with a carbon chain length 11.6
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
0.53 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
frond number
Remarks on result:
other: 0.37-0.75
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
2.63 mg/L
Basis for effect:
frond number
Remarks on result:
other: EC50 was normalized to reflect the toxicity of LAS with a carbon chain length 11.6
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
2.29 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
frond number
Remarks on result:
other: 1.9-2.7
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
4.13 mg/L
Basis for effect:
biomass
Remarks on result:
other: EC50 was normalized to reflect the toxicity of LAS with a carbon chain length 11.6
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
5.63 mg/L
Basis for effect:
other: root length
Remarks on result:
other: EC50 was normalized to reflect the toxicity of LAS with a carbon chain length 11.6
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
5.52 mg/L
Basis for effect:
growth rate
Remarks on result:
other: EC50 was normalized to reflect the toxicity of LAS with a carbon chain length 11.6
Details on results:
These experimental results were normalized to reflect a C11.6 chain length using a highly localized LAS-specific QSAR (Belanger et al, 2016). Toxicity normalization was performed using this basic fo rmula:

ChVc11.6norm = ChVx * (ChVc11.6pred / ChVxpred)

Where ChV is any chronic endpoint result; such as NOEC, LOEC, EC10, etc., ChVc11.6norm is the normalized ChV for the test material,
ChVx is the experimental ChV for the test material,
ChVc11.6pred is the QSAR predicted ChV for the target material with a chain length of C11.6, ChVxpred is the QSAR predicted ChV for a material with the same chain length as the test
material.

A QMRF is available on the QSAR (Belanger et al, 2016) used to provide the predicted results for normalization. More details on this approach are available in the endpoint summary.

- Any visual signs of phytotoxicity (abnormalities): Not reported
- Decrease in frond size: Not reported
- Necrosis / chlorosis: Not reported
- Sinking of fronds: Not reported
- Other: Frond count decreased dose dependently from Day 4 with increase in exposure concentration
- Any stimulation of growth found in any treatment: Not reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not reported
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Endpoint values for frond count were derived using SAS v. 9.1.3. The frond count data were fit to an empirical non-linear model. The growth rate data were fit to empirical models derived directly from the frond count model. Root length and dry weight were fit to the same exponential model given by growth rate. See Appendix to study report for more details on statistical evaluations.

None

Validity criteria fulfilled:
yes
Conclusions:
The 7d EC10 of C11.8 LAS (linear alkylbenzene sulfonate) to Lemna minor was 0.24 mg/L, based on frond count in a flow through test. The result is based on measured concentration. This result is normalized to reflect the toxicity of an LAS with a carbon chain length of 11.6. The EC10, based on frond number is 0.24 mg/L.
Executive summary:

A growth inhibition test inLemna minor(duckweed), following OECD Guideline 221, was conducted on C10-13 LAS, sodium salt (average chain length C11.8). Endpoints included frond count, dry weight, growth rate and root length after a 7 d exposure period in a flow through system. The test concentrations were 0, 2.1, 3.8, 8, 17 and 34 mg/L (measured). There were 4 replicates at each test concentration. The EC10 value, based on frond number, was 0.24 mg/L. The EC50 value, also based on frond number, was 2.63 mg/L. These results are normalized to reflect the toxicity of a LAS with a carbon chain length of 11.6 (Verge, 1996). The non-normalized results are 0.21 and 2.29 mg/L, respectively (Bishop, 1980 and 1981). 

Description of key information

Study 1 (C10-13 LAS):

The long-term toxicity of C10-13 LAS, sodium salt (average chain length C11.6) to aquatic plants (Elodea canadensis) was evaluated in a 28 d model ecosystem flow-through test. The nominal test concentrations were 0, 0.5, 1.0, 2.0 and 4.0 mg/L, confirmed by analytical measurements. The growth and productivity of Elodea was recorded after the exposure period of 28 d. Growth inhibition was not observed even at highest tested concentration (4 mg/L). Growth throughout the exposure period approximately doubled the initial biomass of the vegetative shoots used at the start of the exposure.  The initial biomass was 37 mg ashed-free dry weight. The overall mean for all treatment concentrations was 72 mg ashed-free dry weight. Overlap of 95% confidence limits for all treatments with unexposed control production confirmed the lack of LAS effects throughout the exposure period. Hence, the NOEC was found to be >4 mg/L (Maki, 1981).

Study 2 (C10-13 LAS):

A growth inhibition test in Lemna minor (duckweed), following OECD Guideline 221, was conducted on C10-13 LAS, sodium salt (average chain length C11.8). Endpoints included frond count, dry weight, growth rate and root length after a 7 d exposure period in a flow through system. The test concentrations were 0, 2.1, 3.8, 8, 17 and 34 mg/L (measured). There were 4 replicates at each test concentration. The EC10 value, based on frond number, was 0.24 mg/L. The EC50 value, also based on frond number, was 2.63 mg/L. These results are normalized to reflect the toxicity of a LAS with a carbon chain length of 11.6 (Verge, 1996). The non-normalized results are 0.21 and 2.29 mg/L, respectively (Bishop, 1980 and 1981). 

Key value for chemical safety assessment

EC10 or NOEC for freshwater plants:
4 mg/L

Additional information

The point of departure for aquatic PNEC derivation is the 56 d NOEC of 0.268 mg/L from a mesocosm model ecosystem (Belanger, 1997, 2002 and 2004; Lowe, 1996).