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Ecotoxicological information

Toxicity to aquatic plants other than algae

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Administrative data

Endpoint:
toxicity to aquatic plants other than algae
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
11/2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Qualifier:
according to
Guideline:
other: Environment Canada (EC). 2007. Biological Test Method: Test for measuring the inhibition of growth using the freshwater macrophyte, Lemna minor. Conservation and protection, Environmental Protection, Ottawa, ON, Report EPS 1/RM/37.
Principles of method if other than guideline:
/
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): Aluminum nitrate nonahydrate
- Molecular formula (if other than submission substance): Al(NO3))3.9H2O
-Source: J.T. Baker, Philipsburg, NJ, USA
- Analytical purity:100.6%
- Impurities (identity and concentrations): Ca = 0.001%, Cl = 0.001%, Mg = 0.0002%, K = 0.0005%, Na = 0.003%, Pb = 0.0006%, Fe = 0.001%
- Purity test date: 20/10/2009
- Lot/batch No.:H43606
- Storage condition of test material:stored and sealed in its original container at room temperature
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
/

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Analytical samples were taken from each treatment
- Sampling method:

TOTAL ALUMINUM
Approximately 5 mL was drawn into the syringe to rinse the inside of the syringe and then expunged. Then 15 mL of sample was drawn into the syringe and injected into a 15 mL polypropylene conical tube. Samples of old waters were taken from a composite of each replicate from each concentration. Old water was poured off from the top half/layer of the water column within each beaker.

DISSOLVED ALUMINUM
Approximately 20 mL was drawn into the syringe of which 5 mL was pushed through the filter to waste and the remaining 15 mL was collected into a 15-mL polypropylene conical test tube.

MONOMERIC ALUMINUM
Following collection of a sample for dissolved aluminum analysis, the same filter was used to filter approximately 15 mL aliquots for monomeric aluminum analysis. Monomeric samples were immediately analyzed.
- Sample storage conditions before analysis: Samples were preserved with trace metal grade nitric acid (AR-ACS grade, Mallinckrodt Chemical, Hazelwood, MO, USA) to pH < 2 and refrigerated (0 - 4 °C) prior to analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:A stock solution of 10 g Al/L was prepared by addition of 69.51 grams of Al(NO3)3·9H2O to 500 mL of Milli-Q water in a volumetric flask. The stock solution was then stored in a plastic container in the dark at 0 - 4°C. All concentrations within this report are expressed as micrograms Al per liter (μg/L Al).

Test organisms

Test organisms (species):
Lemna minor
Details on test organisms:
TEST ORGANISM
- Common name:Common Duckweed
- Source (laboratory, culture collection):OSU AquaTox (An initial stock of L. minor fronds was obtained from the Canadian Phycological Culture Centre (Waterloo, Ontario, Canada))
- Age of inoculum (at test initiation):10 days
- Method of cultivation:in Swedish Standard (SIS) Growth Media (OECD 2006)

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Post exposure observation period:
/

Test conditions

Hardness:
32 mg/L as CaCO3
Test temperature:
25 +/- 2°C
pH:
5.8-6.3
Dissolved oxygen:
/
Salinity:
/
Nominal and measured concentrations:
Nominal concentration: 0, 625, 1250, 2500, 5000 and 10000μg/L
Average measured total Al concentration: <15.1; 666.8; 1116.2; 2760.3; 5313.8; 10482.3 μg/L
Details on test conditions:
TEST SYSTEM
- Incubation chamber used: yes
- Test vessel:250ml glass beaker
- Material, size, headspace, fill volume: glass beaker filled with 200ml of test solution
- Renewal rate of test solution (frequency/flow rate): water changes were performed on day 2 and 5
- Control end cells density:
- No. of colonies per vessel: 9-12
- No. of fronds per colony:2-4
- No. of vessels per concentration (replicates):3
- No. of vessels per control (replicates):1

GROWTH MEDIUM
- Detailed composition if non-standard medium was used: Swedish Standard Growth Media with the addition of MOPS (4-morpholinepropane sulphonic acid). The media was prepared as detailed in standard OECD methods (OECD 2006). The water was pH adjusted to 6.0 using dilute HCl and NaOH.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution/control water used in the toxicity test was Swedish Standard Growth Media with the addition of MOPS (4-morpholinepropane sulphonic acid). The media was prepared as detailed in standard OECD methods (OECD 2006). The water was pH adjusted to 6.0 using dilute HCl and NaOH. A new batch of media was prepared on the day prior to test initiation and each water renewal. At test initiation, the dilution/control water was characterized for hardness, alkalinity, and ammonia (NH3). The water was warmed to test temperature (25 ± 2°C) prior to use in the toxicity tests.
- Ca/mg ratio:1.17
- Alkalinity: 16 mg/L as CaCO3
- DOC: 208 mg/L
- Total residual chlorine: N/A
- Ammonia: <1.0 mg/L
- Calcium: 8.65 mg/L
- Magnesium: 7.42 mg/L
- Sodium: 28.2 mg/L
- Potassium: 3.34 mg/L
- Chloride: 19.9 mg/L
- Sulfate: 25.6 mg/L
- Culture medium different from test medium:no
- Intervals of water quality measurement: at test initiation, at renewal, (in both freshly prepared renewal waters and discarded waters), and at test termination. Hardness, alkalinity, and total ammonia were measured in the dilution water at test initiation.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Photoperiod:under a continuous light cycle
- Light intensity and quality: cool-white fluorescent lights at 600-930 foot candles

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number:[manual counting
- Determination of biomass:dry weight

Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
2 760.3 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Remarks:
: total Al
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
2 760.3 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Remarks:
: total Al
Basis for effect:
other: dry weight
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
2 760.3 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Remarks:
: total Al
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
5 313.8 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Remarks:
: total Al
Basis for effect:
frond number
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
5 313.8 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Remarks:
: total Al
Basis for effect:
other: dry weight
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
5 313.8 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Remarks:
: total Al
Basis for effect:
growth rate
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
2 694 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Remarks:
: total Al
Basis for effect:
frond number
Remarks on result:
other: CL: 1308-5550 μg/L
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
2 175 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Remarks:
: total Al
Basis for effect:
other: dry weight
Remarks on result:
other: CL: 417-11352 μg/L
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
4 545 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Remarks:
: total Al
Basis for effect:
growth rate
Remarks on result:
other: CL: 3348-6170 μg/L
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
3 982 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Remarks:
: total Al
Basis for effect:
frond number
Remarks on result:
other: CL: 2346-6757 μg/L
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
4 093 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Remarks:
: total Al
Basis for effect:
other: dry weight
Remarks on result:
other: CL: 1350 - 12413 μg/L
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
6 924 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Remarks:
: total Al
Basis for effect:
growth rate
Remarks on result:
other: CL: 5683-8437 μg/L
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
8 643 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Remarks:
: total Al
Basis for effect:
frond number
Remarks on result:
other: CL: 6800-10985 μg/L
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
14 537 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Remarks:
: total Al
Basis for effect:
other: dry weight
Remarks on result:
other: CL: 7063-29185 μg/L
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
15 966 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
element
Remarks:
: total Al
Basis for effect:
growth rate
Remarks on result:
other: CL: 13012 - 19590 μg/L
Details on results:
/
Results with reference substance (positive control):
/
Reported statistics and error estimates:
STATISTICAL ANALYSIS
Statistical analysis was performed using measured average total Al concentrations of newly prepared solutions. Differences in the number of fronds, dry weight, and growth rate at test termination were evaluated using a statistical computer package (Comprehensive Environmental Toxicity Information System [CETIS], Tidepool Scientific Software, McKinleyville, CA, USA and Toxicity Relationship Analysis Program [TRAP], Duluth, MN, USA). If the data met the assumptions of normality and homogeneity, the NOEC and LOEC were estimated using an analysis of variance to compare (p = 0.05) organism performance in the experimental treatments with that observed in the control. The effective concentrations to reduce growth by 10%, 20%, or 50% relative to control performance (EC10/EC20/EC50) were estimated using threshold sigmoid regression analysis. Exposure concentrations were log-transformed before determination of the EC10, EC20, and EC50 values.

Any other information on results incl. tables

Table: Biological results

 Average measured total concentration Al (μg/L)  Frond Count  Total dry weight (mg)  Growth rate (units)
 2.0  137.0 ± 13.9  14.58 ± 2.22  0.3885 ± 0.014
 666.8  137.7 ± 21.0  14.23 ± 1.68  0.3885 ± 0.022
 1116.2  118.3 ± 11.0  13.79 ± 1.67  0.3676 ± 0.013
 2760.3  125.0 ± 11.8  13.20 ± 0.98  0.3755 ± 0.013
 5313.8  89.7 ± 3.8 10.29 ± 0.80   0.3283 ± 0.006
 10482.3  53.3 ± 2.5  8.95 ± 1.15  0.2541 ± 0.007

Applicant's summary and conclusion

Conclusions:
Total measured exposure concentrations ranged from 2 to 10,482.3 μg Al/L and resulted in a significant effect on plant growth at the highest two treatments. The study resulted in a no observable effect concentration [NOEC] of 2760.3 μg/L total Al and a lowest observable effect concentration [LOEC] of 5313.8 μg/L total Al for growth. Total dry weight was determined to be the most sensitive growth endpoint. The effective concentrations to reduce growth (as total dry weight) by 10% and 20% relative to control performance (EC10 and EC20 with 95% confidence intervals) were 2175 (417 – 11352) and 4093 (1350 – 12413) μg/L total Al, respectively.
Executive summary:

As part of an environmental program designed to provide data for the setting of water quality standards, data describing the chronic toxicity of aluminum to a variety of aquatic organisms are needed. Aluminum toxicity is a function of the chemical species of aluminum present in the water and this speciation is a function of the physico/chemical properties (e.g., pH) of the water. To this end, efforts are underway to develop data describing the chronic toxicity of aluminum to aquatic life at hydrogen ion concentrations (i.e., pH) typical of natural environmental conditions (i.e., pH of 6.0). The study reported herein determines the chronic toxicity of aluminum, at a pH of 6.0, to the freshwater duckweed, Lemna minor. Use of this test organism qualifies as a level of organization (Phylum) that is not currently represented as part of the minimum taxonomic requirements for calculation of a predicted no effect concentration (PNEC) for the freshwater aquatic compartment, within the context of a species sensitivity distribution approach (European Commission 2003). In this study, L. minor was exposed to a series of aluminum concentrations for seven days, starting as 10 day old plants. To allow for possible changes in aluminum speciation, exposure solutions were aged for a 3- hour equilibration period prior to plant exposure. Nominal test concentrations ranged from 0 to 10,000 μg Al/L and total, dissolved, and monomeric aluminum were measured throughout the test. As the formation of insoluble chemical species was apparent through low dissolved measurements (all concentrations measuring below 15.1 μg Al/L, with two exceptions [21.8 and 25.8 μg Al/L]), total Al was used to interpret the biological data in this study. Measured pH values in the test averaged a pH of 6.1. Total measured exposure concentrations ranged from 2 to 10,482.3 μg Al/L and resulted in a significant effect on plant growth at the highest two treatments. The study resulted in a no observable effect concentration [NOEC] of 2760.3 μg/L total Al and a lowest observable effect concentration [LOEC] of 5313.8 μg/L total Al for growth. Total dry weight was determined to be the most sensitive growth endpoint. The effective concentrations to reduce growth (as total dry weight) by 10% and 20% relative to control performance (EC10 and EC20 with 95% confidence intervals) were 2175 (417 – 11352) and 4093 (1350 – 12413) μg/L total Al, respectively.