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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
June 30 to July 28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1350 (Mysid Chronic Toxicity Test)
Qualifier:
according to guideline
Guideline:
other: ASTM E1191, and U.S. EPA FIFRA Subdivision E, Section 72-4
Principles of method if other than guideline:
salinity 20‰
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The concentrations of boron in test solutions were determined in samples collected two days prior to initiation (day –2) and on study days 0, 7, 14, 21, and 28 (termination) of the definitive test. A single replicate sample was collected from the dilution-water control, and test-substance treatments; alternating replicates were sampled at each sampling event. The concentration of boron in the diluter stock solutions were determined in samples collected on the same days as the test solutions.

A 10-mL sample was collected from the dilution-water control, each test-substance treatment, and from the diluter stock solution. QC samples were prepared by fortifying water collected from the dilution-water control with boric acid at concentrations of 1.82 and 38.7 mg B/L. From each 10-mL sample, a 0.050-mL volume was removed from all but the highest test substance treatment and highest QC sample, and diluted to a 10-mL volume with ABC reagent water. A 0.025-mL volume was removed from each 10-mL sample of the highest test substance treatment and highest QC sample, and diluted to a 10-mL volume with ABC reagent water. A 0.025-mL volume of the diluted-diluter stock solution sample was further diluted with ABC reagent water to a volume of 10 mL. The standard curve range for these analyses was 0.000 to 0.140 mg B/L.
Vehicle:
no
Details on test solutions:
Diluter stock solutions were prepared at a target concentration of 6,210 mg B/L by diluting approximately 35.506 g (6.210 g as boron corrected for boron content in boric acid) of boric acid per liter of saltwater. Diluter stock solutions volumes of 6 or 18 L were prepared in this manner on June 25 as well as July 2, 13, 20 and 26, 2010. All diluter stock solutions were stored at room temperature and shielded from light.
Test organisms (species):
Americamysis bahia (previous name: Mysidopsis bahia)
Details on test organisms:
Mysid shrimp (Americamysis bahia) were obtained from in-house cultures that were maintained at ABC Laboratories, Inc. The initial parental mysids were obtained from Aquatic Bio Systems Inc. on October 29, 2008, and May 27, 2010. The organisms from Aquatic Bio Systems Inc. were received with documentation stating them to be Americamysis bahia. The adult mysids used to generate the juvenile test organisms were cultured in artificial saltwater with salinity of approximately 20‰ and at a temperature of approximately 25°C. The animals were fed brine shrimp nauplii (Artemia sp. <48 hours old). Periodic lots of feed were screened for contaminants. Records of feed suppliers and the results of the contaminant analyses are on file at ABC Laboratories, Inc. A record of mysid observations and juvenile production numbers during the culture period was maintained.
Juvenile mysids were collected on June 30, 2010 and were <24 hours old at test initiation. The mysids were fed live brine shrimp nauplii (Artemia sp.) at least two times daily during the definitive test period.

Mysids were fed ad libitum brine shrimp nauplii (Artemia sp.; 24-48 hours old) at least two times daily. The concentration of the nauplii in the food stock was determined by replicate counts of aliquots pipetted onto filter paper. The food stock concentration ranged from 280 to 870 brine shrimp/mL (mean ± standard deviation [SD]: 626 ± 145 brine shrimp/mL). The food stock was enriched the day of use with a mixture of Salt Creek HUFA Enrich, AlgaMac-3000, and AlgaMac-Enhance, all of which are commercially available components. All control- and test- substance treatments received, as near as reasonably possible, the same ration (i.e., volume) of food. The enrichment mixture was prepared by diluting 1 mL of Salt Creek HUFA Enrich and 1/8 teaspoon of an approximate 40:60 AlgaMac-3000:AlgaMac Enhance mixture in a household blender with 200 mL saltwater and then blended for approximately one minute. The enrichment mixture was then added to the food stock at the rate of 50 mL/L of food stock.

The body lengths of mysids (as measured by total midline body length) were measured to the nearest 0.1 mm with a dissecting microscope.



Test type:
flow-through
Water media type:
saltwater
Limit test:
no
Total exposure duration:
28 d
Test temperature:
24.2 to 25.4°C
pH:
7.68 to 8.11
Dissolved oxygen:
6.25 to 7.40 mg/L (87 to 103% saturation)
Salinity:
18.9 to 19.9 ‰
Nominal and measured concentrations:
Nominal concentrations: 0 (dilution-water control), 2.0, 4.0, 8.0, 16, and 32 mg B/L.
Mean calculated concentrations of total boron: 3.25 (dilution water control), 5.34, 7.66, 11.8, 22.0, and 36.4 mg B/L
Mean measured concentrations as added boron: 0.0 (dilution-water control), 1.96, 4.28, 8.38, 18.6, and 33.1 mg B/L
Details on test conditions:
The dilution water was a laboratory saltwater prepared by adding a commercial sea salt mix (Crystal Sea Marine Mix; Marine Enterprises International, Inc. Baltimore, Maryland) to laboratory freshwater at a target salinity 20 ± 2‰. The laboratory freshwater consists of well water blended with well water that was demineralized by reverse osmosis to yield water with a total hardness ranging from 130 to 160 mg CaCO3/L. The dilution water was heated and aerated prior to its delivery into the diluter system. As the dilution water entered the diluter system, it passed through a particulate filter and an ultraviolet sterilizer.

During the definitive test, test solutions were delivered to three replicate test chambers and each replicate test chamber received approximately 0.67 L at each diluter cycle. The accuracy of the delivery of the test solution to the test chambers was verified by volumetric measurement before test initiation and adjusted such that the delivered volume was ±10% of the target volume.

Each test chamber used for the definitive test measured approximately 22 cm wide, 38 cm long, and 30 cm high. Each definitive-test chamber had a glass pane in the middle of the tank containing two holes near the bottom of the tank. This partition effectively cut the test chamber volume in half when the holes were stopped with silicone stoppers. The greatest depth of the test solution was approximately 12 cm, which yielded a maximum volume of approximately 10 L when one side was in use and approximately 20 L when both sides of the test chamber were in use. All test chambers were individually drained through the side of the chamber to a floor drain.

Mysids were maintained within retention baskets (constructed from nylon screening) to facilitate daily observations and enumeration. Because retention baskets were not completely isolated from one another, these retention baskets were not replicates. Only the test chamber containing the retention baskets was a true replicate.

Observations of mortality and sublethal responses F0-mysid generation were made daily for the duration of the testing period. Dead mysids were counted, differentiated by gender (if mature), and removed daily. Any missing mysids at the time of observation were noted as not found and were considered dead if not observed on following days; that mortality was subsequently reflected back to the time that the mysid was first observed missing. Any mysids that were inadvertently damaged or died as a result of becoming impinged onto the retention- or brood-basket mesh were removed from the initial number of mysids for those replicates and were not considered to be treatment-related mortality.
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
33.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
mortality
Remarks:
F0 mysids
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
33.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
growth
Remarks:
length of F0-male mysids
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
33.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
growth
Remarks:
length of F0-female mysids
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
18.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
reproduction
Remarks:
mean total young per F0-female
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
33.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
reproduction
Remarks:
mean total young per F0-female
Remarks on result:
other: added boron
Duration:
11 d
Dose descriptor:
NOEC
Effect conc.:
33.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
mortality
Remarks:
of F1 mysids
Remarks on result:
other: added boron
Duration:
12 d
Dose descriptor:
NOEC
Effect conc.:
33.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
growth
Remarks:
length of F1-male mysids
Remarks on result:
other: added boron
Duration:
12 d
Dose descriptor:
NOEC
Effect conc.:
33.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
growth
Remarks:
length of F1-female mysids
Remarks on result:
other: added boron
Reported statistics and error estimates:
Statistical analysis based on one-way ANOVA followed by Dunnett's test. ECx estimates obtained from probit method and trimmed Spearman-Karber method if poor fit in the probit approach.
Validity criteria fulfilled:
yes
Remarks:
The water-quality characteristics remained within the tolerance limits set forth in the protocol.
Conclusions:
The test acceptability criteria for this study were met. The water-quality characteristics remained within the tolerance limits set forth in the protocol. Survival of the dilution-water control F0 mysids was >90%. The percentage of dilution-water control F0-female mysids available to produce young that actually did produce a brood was 100%. The average total number of young produced per dilution-water control F0-female mysid was 36.6.
Based on mean total measured concentrations of boron during the 28-day exposure, the day-28 F0-mysid survival NOEC was 36.4 mg B/L, the highest boron concentration tested. The NOEC for F1-mysid survival on day 9 was 36.4 mg B/L. Mean total young per F0 female was the most sensitive endpoint and the NOEC for this parameter was 22.0 mg B/L. An EC10 could be computed for only one endpoint, mean total young per F0-female mysid, and was 17.5 mg B/L.
If the endpoints are stated based on added boron (total boron minus measured concentration natural boron in the control), then the NOECadded boron for the most sensitive endpoint was 18.6 mg Badded/L for the mean total young per F0 female. The NOEC for all other endpoints was 33.1 mg Badded/L.
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
other: OECD guideline 219
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Analytical measurements were made on additional replicate systems at day 0 and day 28 for determination of test substance concentrations in overlying water.
Vehicle:
no
Test organisms (species):
other: Chironomus riparius
Details on test organisms:
Laboratory cultures used to provide 1st instar (1-day old) individuals at test initiation. Age was confirmed by measurement of head capsule widths of a representative sample.

Breeding conditions: Cultured at room temperature (21-25 degrees C) in laboratory culture water.
Handling of egg masses and larvae: Egg masses collected and isolated in freshwater, maintained at 20 degrees C.
- Feeding during test: prepared invertebrate food- a suspension of commercial fish flake food
- Amount: Daily, 2.5-3 ml of 2.0 mg/ml during 1st 10 days, 6-8 ml for remainder
Test type:
static
Water media type:
freshwater
Total exposure duration:
28 d
Hardness:
140 to 162 mg CaCO3/L
Test temperature:
19.4 to 21.7 ° C
pH:
8.5 to 8.7
Dissolved oxygen:
5.2 to 9.3 mg/L (60 to 95% saturation)
Nominal and measured concentrations:
Nominal concentrations definitive test : 0 (control), 2.5, 5.0, 10, 20, 40 and 80 mg B/L.
Geometric Mean Measured Overlying Water Total Concentrations: 0.298, 3.06, 5.93, 11.0, 20.4, 43.3, and 89.3 mg B/L
Geometric Mean Measured Overlying Water Added Concentrations: 0, 2.76, 5.63, 10.7, 20.1, 43.0, and 89.0 mg B/L
Details on test conditions:
Test chambers were 1-L glass jars containing approximately 255 g of sediment (clean washed sand) Sediment depth was approximately 2 cm. Dilution water (600 mL) was added to each replicate chamber using a deflector to minimize disturbance of the sediment. Water depth was approx 8 cm, so the water: sediment volume ratio was approx 4:1. Polypropylene emergence traps with nylon mesh were used to monitor adult midge emergence. Chambers were left for 1 d to equilibrate before the test started.

At least 7 replicate chambers were prepared for each treatment level and control. Two were used to provide samples for chemical analysis and 3 chambers were used for biological observations. 20 midge larvae were added to each chamber for the chemical and biological observations. Seven different exposures were tested, a control and six treatments. Larvae were impartially added to cups, then the cups randomly assigned to a replicate chamber.

Overlying water was aerated during the test beginning 24 h after addition of the organisms by gentle bubbling. Midges were fed 2.5 to 3 ml of a commercial flake fish food suspension (2.0 mg/ml) for the first 10 d, then 8 ml daily for the remainder of the test. Algae were added during the first 3 days as food for the first instar larvae. Lighting was 16 h: 8 h light:dark cycle with a 30 m transition period. Light intensity was 520 lux. Temperature was maintained at 20 +/- 2 degrees C by keeping the test chambers in a flowing water bath. Water was not replaced during the test.

Emergence of adult flies was monitored from day 18. Presence of adults or exuviae in traps was monitored. Adult flies were identified by gender.
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
20.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
LC10
Effect conc.:
52.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
mortality
Remarks on result:
other: confidence limits: 49.8 - 54.6 mg/L
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
20.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
other: emergence
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
43.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
other: emergence time
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
43.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
other: development rate
Details on results:
The nominal concentrations were added in the effect concentrations table, following the usual practice that a nominal concentration may be used if the measured values are reasonably close to the nominal. EC10 could not be calculated for the endpoints emergence time and development rate. For the endpoint mortality both NOEC and LC10 values are reported, and preference is given to the use of LC10 value for PNEC derivation. For the endpoint emergence, an EC10 of 9.7 mg/L (7.4 E-23 - 1.3 E+24 mg/L) has been calculated but was not fruther used because of the large confidence limits.
Results with reference substance (positive control):
s
Reported statistics and error estimates:
Statistical analysis based on one-way ANOVA followed by Dunnett's test. The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A.
Validity criteria fulfilled:
yes
Conclusions:
Expressed as nominal added boron the NOEC values for survival and percent emergence were 20 mg B-added/L and LOECs were 40 mg B-added/L. The NOEC value for development rate was 40 mg B-added/L.

Based on survival and percent emergence, the NOEC (total) was 20.4 mg B/L and LOEC (total) was 43.3 mg B/L. The EC10 (total) for survival was 43.0 mg B/L. The EC10 (total) for percent emergence was 40.8 mg B/L (95% CI 30.5 to 51.1 mg B/L) The NOECs (total) for time to emergence for males and females (calculated separately and combined) were 43.3 mg B/L. The NOECs (total) for development rate for males and females (calculated separately and combined) were 43.3 mg B/L.

Expressed as added boron the NOEC values for survival and percent emergence were 20.1 mg B-added/L and LOECs were 43.0 mg B-added/L. The NOEC value for development rate was 43.0 mg B-added/L. The EC10 for survival would be 42.7 mg B-added/L and the EC10 for percent emergence would be 40.5 mg B-added/L.

Analyses of the overlying water at test initiation and termination documented that the boron concentrations were relatively constant in the overlying water. This suggests that the silica sand sediment did not remove any significant amount of the boric acid spiked into the water.
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to Guideline study. Pre dates OECD Guideline
Qualifier:
equivalent or similar to guideline
Guideline:
other: ASTM Subcommittee on Safety to Aquatic Organisms : Proposed standard practise for conduction renewal life-cycle toxicity tests with Daphnia magna draft nr2 1982
GLP compliance:
not specified
Analytical monitoring:
yes
Vehicle:
no
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnid
- Source: Brood stock
- Age at study initiation (mean and range, SD): < 24h
- Method of breeding: The brood stock was cultured and maintained in aerated (90-105% saturation) 2.91 - liter animal jars. A 2.0-liter open-ended tube with 1.0 mm nylon bolting cloth affixed to the bottom was contained within each jar to facilitate harvesting first instars. Th screened end of the tube was held approximately 1.5 cm off the bottom by two Pyrex triangles placed one on top of the other. Each tube contained 25 adult daphnids. When the tube was lifted, the adults remained on the screen while the first instars passed into the beaker for harvesting. Each brood stock animal jar contained the appropriate amount of food and water made up to a 2.0 liter volume. The brood stock food, water and glass receptacles were changed 3 time per week. The brood stock was kept in an environmental chamber set at 20 +-1°C and a photoperiod of 16h light : 8h dark. Twenty-four hours before testing, mutiparous females were isolated. The neonates produced by these adults were removed and used for testing within 24h.
- Feeding during test
- Food type: S. capricornutum
- Amount: 1.25 mg/L dry weight
- Frequency: 3 days per week
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
170 mg/l (as CaCO3)
Test temperature:
19.5 - 20.5 °C
Dissolved oxygen:
7.3 - 8.0 mg/L
Nominal and measured concentrations:
The nominal concentrations were 7. 14. 28. 56 and 105 mg/L as boron
Measured concentrations : 6.4, 13.6, 29.4, 59.3 mg/L as boron
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 600ml Pyrex beakers. Each beaker contained a 1.0 mm mesh stainless steel platform and 5 glass tubes (2.5 x 12.5 cm) with 363 µm nylon mesh bottoms. The tubes were placed on the platform to allow water circulation. Each test beaker contained 500 ml of the appropriate amounts of test material, food and water.
- Aeration: gently aerated to achieve 90 to 105 % saturation.
- Renewal rate of test solution (frequency/flow rate): 3 days per week
- No. of organisms per vessel: 5 (1 per tube)
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Lake Huron water was used to culture daphnids and as the test dilution water. This water was carbon-filtered, UV-irradiated and adjusted to a hardness of approximately 170 mg/L as (CaCO3) with CaCl2. The water was then autoclaved at 120°C and 124.1 kPa for 35 min.
- Alkalinity:58 +- 5 mg/L as CaCO3
- Conductivity: 290 +- 21 µmhos/cm


OTHER TEST CONDITIONS
- Photoperiod: 16 h light: 8 h dark


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 3 days per week the young produced by each adult were counted and discarded and adult survival was recorded.
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
29.4 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
6.4 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
6.4 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
growth
Remarks:
mean size
Duration:
21 d
Dose descriptor:
LC10
Effect conc.:
41.1 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
mortality
Remarks on result:
other: confidence limits: 8.9 - 189.3 mg/L
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
6.6 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
reproduction
Remarks on result:
other: confidence limits: 0.6 - 72 mg/L
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
29.2 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
growth
Remarks on result:
other: confidence limits: 17.3 - 49.3 mg/L
Details on results:
NOEC and LC10/EC10 values are provided for the endpoints reported, i.e. mortality, reproduction and growth. Preference is given to the use of EC10/LC10 values for PNEC derivation
Reported statistics and error estimates:
The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A. Finney's method of probit analysis or the moving average method was used to calculate the LC50 values. Data were analyzed for NOEC and LOEC using a two tailed Dunnett's t test (alpha = 0.05). Mean comparisons between test and control concentrations were performed on the following endpoints : mean number of broods per daphnid, mean total young per daphnid and mean length.
Duration Endpoint Effect conc. Nominal/Measured Conc. based on Basis for effect Remarks (e.g. 95% CL)
21 d LC50 52.2 mg/L measured element mortality
21 d LOEC 59.3 mg/L measured element mortality
21 d LOEC 13.6 mg/L measured element reproduction
 21 d LOEC   13.6 mg/L  measured element   growth (mean size)  
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Qualifier:
according to guideline
Guideline:
other: EU New Draft Guideline "Prolonged toxicity study with Daphnia magna (inhibition of reproduction)"
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The samples of approximately 50 ml were received in polyethylene bottles from the Department of Environmental Toxicology of the TNO Nutrition and Food Research Institute on 5 August and 20 October 1999. Upon receipt they were identified with the unique codes. They were analysed on 11 August and 22 November 1999.
Vehicle:
no
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Source: fresh-water crustacean Daphnia magna, cultrued in the laboratory under standard conditions. Daphnia magna has been cultured at TNO in the dilution water used for the present test
- Feeding during test
- Food type: cells of the unicellular alga Chlorella sp. and some 'yeast' was added as extra food
- Frequency: daily

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
212 mg/L (as CaCO3)
Test temperature:
19.3 - 20.9 °C
pH:
7.2 - 8.0
Dissolved oxygen:
> 7.4 mg/L
Nominal and measured concentrations:
Nominal concentration : 1.8, 3.2, 5.6, 10, 18, 32 and 56 mg B/L.
Measured concentrations: 0.57, 2.59, 3.92, 6.55, 10.78, 19.49, 34.17 and 59.14 mg/L
The actual measured boron concentrations were between 98% and 102% of the nominal values in the newly prepared test solutions and between 105 and 108% in the spent solutions
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 100 ml all-glass beakers, each containing 50 ml of exposure medium.
- Aeration: no aeration
- Renewal rate of test solution (frequency/flow rate): every Monday, Wednesday and Friday
- No. of organisms per vessel: 1


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: DSWL-E prepared from ground water
- Total organic carbon: 1.6 mg/L
- Chlorine: 3.5 mmol/L
- Ca/mg ratio: 2 : 1
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH values and oxygen concentrations were measured weekly in one of the newly prepared control test vessels and in one vessel of each test substance concentration. The pH and oxygen concentration were also measured in the same spent solutions, just before replacement and in any test vessel with a dead animal. The pH of the exposure media was not adjusted to that of the dilution water. The temperature was measured in one of the control vessels and the highest concentration tested just before and just after replacement of the test media.


OTHER TEST CONDITIONS
- Photoperiod: 16 light - 8 dark with transition periods of 30 min


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The dead animals were recorded daily, and removed if applicable, and any young born were counted and removed at each replacement time and at the end of the test. The condition (swimming behaviour, colour or any other visually observable morphological or behavioural criterion) and the size of the original test animals were qualitatively compared with those of the control animals at the same time.


RANGE-FINDING STUDY
- Appropriate concentrations for the toxicity tests were determined in a preliminary range finding test.
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
10.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
17.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
reproduction
Remarks on result:
other: confidence limits: 15.5 - 20.2 mg/L
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
34.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
mortality
Details on results:
NOEC and LC10 values have been calculated for the endpoint reproduction. Preference is given to the use of the EC10 (17.7 mg/L) compared to the NOEC (10.8 mg/L ) value for reporduction. For the endpoint mortality, both the NOEC (34.2 mg/L) and LC10 (44.5 mg/L) values could be calculated. However the LC10 for the endpoint mortality was not reliable because of the large confidence interval between 0 mg/L - infinity.
Reported statistics and error estimates:
Statistical significance for mortality was determined with a binomial test at a 95 % and 99 % significance level. Statistical significance for reproduction was dermined using the two-tailed Dunnett-test at a 95 % and 99 % significance level using the mean number of young per female as observed values. In both cases, the obersvations at each concentrion were compared with those of the control. In the case of significance at the 99% level only that significance is given.

The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A

If the reproduction of Daphnia magna is inhibited at sublethal concentrations the (cumulative) reproduction can often be described by: Ri(c) = R(0)/[1 + (c/EC50)^b] + ei in which Ri is the (cumulative) reproduction in the ith test vessel. R depends on the concentration of the test compound c. EC50 is the concentration at which the reproduction is reduced to 50% of the reproduction in the unstressed control. b is a slope parameter ( the larger b, the steeper the slope of the relation between concentration and reproduction). In each experiment a certain error, ei, will occur. These errors are assumed to be independent and normal distributed with expectation 0 and variance σ². Given these assumptions about the relation between concentration and reproduction and about the error in the observations the maximum likelihood estimations of the parameters of the function are calculated. Estimation of the LC50 and its confidence interval : At a given time, the mortality probability of an individual is assumed to be logistically related to the logarithm of the concentration, i.e. pi = (ei+p0)/(1+ei), where ei = (ci/α)^(1/β) and pi is the mortality probability in the ith concentration p0 is the mortality probability in concentration α is the LC50 β is a parameter inversely proportional to the maximum gradient of the dose response function ci is the ith concentration The parameters p0, α and β are estimated from the counts by means of the maximum likelihood method; i.e. the parameter values to be selected maximize the probablity of the counts as a function of the three parameters. Since the distribution of α will not be symmetrical the variance-covariance matrix is not estimated for the parameters p0, α and β themselves, but for p0, γ = ln α and β. The variance-covariance matrix is estimated by the inverse of the information matrix. The 95% confidence interval of the LC50 is now given as. α.exp(+-2[var(γ)]^(1/2))= α.exp(+-2[var(ln(α))]^(1/2))

Duration Endpoint Effect conc. Nominal/Measured Conc. based on Basis for effect Remarks (e.g. 95% CL)
21 d EC50 22 mg/L nominal element reproduction
21 d LOEC 18 mg/L nominal element reproduction
21 d LC50 34 mg/L nominal element mortality
21 d LOEC 56 mg/L nominal element mortality
 21 d LOEC  32 mg/L nominal  element  condition  
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study; well performed study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
other: US EPA 2000 Methods for assessing the toxicity and bioaccumulation of sediment-associated contaminants with freshwater invertebrates. 2nd ed. EPA/600/R-99/064.
GLP compliance:
no
Remarks:
study is not in compliance with GLP
Analytical monitoring:
yes
Details on sampling:
Each treatment sampled at beginning and end of test
Vehicle:
no
Test organisms (species):
other aquatic crustacea: Hyalella azteca
Details on test organisms:
TEST ORGANISM
- Common name: Scud
- Strain/clone:
- Justification for species prescribed by test guideline:
- Source: In-house culture
- Age of parental stock (mean and range, SD):
- Feeding during test: tetramin slurry and maple leaf strip
- Food type: Tetramin and maple leaves
- Amount: tetramin: 200 ul of 5 g/L suspension(blended). Maple leaf at all times.
- Frequency: Every changeover of media (3-4 days)

ACCLIMATION
- Acclimation period:
- Acclimation conditions (same as test or not):
- Type and amount of food:
- Feeding frequency:
- Health during acclimation (any mortality observed):

QUARANTINE (wild caught)
- Duration:
- Health/mortality:

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Test type:
semi-static
Water media type:
freshwater
Total exposure duration:
42 d
Hardness:
102 to 110
Test temperature:
22.1 to 23.8
pH:
7.5 to 8.1
Dissolved oxygen:
6.6 to 8 mg/L
Nominal and measured concentrations:
Nominal: control, 3.13, 6.25, 12.5, 25, 50 mg B/L
Measured: 0.07, 3.2, 6.6, 13.0, 25.9, 51.1 mg B/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 300 ml high form beaker
- Type: open
Material, size, headspace, fill volume: 200 ml
- Aeration: no
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4 at beginning. 3 at end (one was dropped)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted freshwater - RMHRW (reformulated moderately hard reconstituted water) per Smith et al 1997
- Total organic carbon: 0 nominal
- Particulate matter: 0 nominal
- Metals:
- Pesticides:
- Chlorine: 0 nominal
- Ca/mg ratio: 5.40 (mass:mass)
- Conductivity:
- Salinity:
- Culture medium different from test medium: cultured in RMHRW
- Intervals of water quality measurement: boron, pH, conductivity, alkalinity, hardness at on "in" and "out" water for each changeover. temperature and D.O. daily

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 L: 8D

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Survival, dry weight, Number of females, number of offspring per female

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
Duration:
42 d
Dose descriptor:
NOEC
Effect conc.:
25.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
mortality
Duration:
42 d
Dose descriptor:
LOEC
Effect conc.:
51.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
mortality
Duration:
42 d
Dose descriptor:
NOEC
Effect conc.:
6.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
reproduction
Remarks:
(number of offspring per female)
Duration:
42 d
Dose descriptor:
LOEC
Effect conc.:
13 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
reproduction
Remarks:
(number of offspring per female)
Duration:
42 d
Dose descriptor:
other: MATC
Effect conc.:
9.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
reproduction
Remarks:
(number of offspring per female)
Duration:
42 d
Dose descriptor:
NOEC
Effect conc.:
>= 25.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Basis for effect:
other: Dry weight after excluding treatments with significantly lower survival than controls
Reported statistics and error estimates:
Data tested for normality using the Shapiro-Wilk’s Test, and homogeneity of variance using Bartlett’s test. One replicate beaker in the control was lost due to technical error, resulting in unequal numbers of replicates so Bonferroni’s test was used to analyze weight and reproduction data, while Fisher’s exact test was used to analyze survival data. LC10/EC10 could not be calculated

Hyalella data (treatment group measured concentrations shown in mg-B/L)

Survival - day 42

Control 3.2 6.6 13 25.9 51.1
treatment mean 90.0% 82.5% 85.0% 72.5% 87.5% 37.5%*
rep. A lost 90.0% 100.0% 60.0% 90.0% 40.0%
rep. B 90.0% 80.0% 80.0% 90.0% 90.0% 40.0%
rep. C 90.0% 80.0% 90.0% 80.0% 100.0% 20.0%
rep. D 90.0% 80.0% 70.0% 60.0% 70.0% 50.0%
 * indicates mean is significantly different from control
           

Number of offspring per female - day 42

Control 3.2 6.6 13 25.9 51.1
treatment mean 3.2 3.1 2.0 0.8* 0* n/a
rep. A lost 4.0 2.2 1.3 0
rep. B 3.6 1.8 1.0 0.5 0
rep. C 2.6 2.3 1.6 1.3 0
rep. D 3.4 4.3 3.0 0.0 0

* indicates mean is significantly different from control

Dry weight - day 42 (mg/individual)

Control 3.2 6.6 13 25.9 51.1
treatment mean 0.33 0.35 0.31 0.33 0.28 n/a
rep. A lost 0.302 0.263 0.402 0.276
rep. B 0.302 0.377 0.265 0.252 0.240
rep. C 0.355 0.407 0.367 0.270 0.264
rep. D 0.346 0.317 0.362 0.403 0.359
 
           
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
June 16, 2010 to July 14, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1350 (Mysid Chronic Toxicity Test)
Qualifier:
according to guideline
Guideline:
other: ASTM E1191, and U.S. EPA FIFRA Subdivision E, Section 72-4
Principles of method if other than guideline:
salinity 8‰
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The concentrations of boron in test solutions were determined in samples collected seven days prior to initiation (day –7) and on study days 0, 7, 14, 21, and 28 (termination) of the definitive test. A single replicate sample was collected from the dilution-water control, and test-substance treatments; alternating replicates were sampled at each sampling event. The concentration of boron in the diluter stock solutions were determined in samples collected on the same days as the test solutions.

A 10-mL sample was collected from the dilution-water control, each test-substance treatment, and from the diluter stock solution. QC samples were prepared by fortifying water collected from the dilution-water control with boric acid at concentrations of 1.82 and 36.0 mg B/L. From each 10-mL sample, a 0.050-mL volume was removed from all but the highest test substance treatment and highest QC sample, and diluted to a 10-mL volume with ABC reagent water. A 0.020-mL volume was removed from each 10-mL sample of the highest test substance treatment and highest QC sample, and diluted to a 10-mL volume with ABC reagent water. A 0.025-mL volume of the diluted-diluter stock solution sample was further diluted with ABC reagent water to a volume of 10 mL. The standard curve range for these analyses was 0.000 to 0.800 mg B/L.
Vehicle:
no
Details on test solutions:
Diluter stock solutions were prepared at a target concentration of 6,140 mg B/L by diluting approximately 35.106 g (6.175 g as boron corrected for boron content in boric acid) of boric acid per liter of saltwater. Diluter stock solutions volumes of 4, 5, or 18 L were prepared in this manner on June 1, 2, 17, and 23 as well as July 2, 7 and 12, 2010. All diluter stock solutions were stored at room temperature and shielded from light.
Test organisms (species):
Americamysis bahia (previous name: Mysidopsis bahia)
Details on test organisms:
Mysid shrimp (Americamysis bahia) were obtained from in-house cultures that were maintained at ABC Laboratories, Inc. The initial parental mysids were obtained from Aquatic Bio Systems Inc. on October 29, 2008, and May 27, 2010. The organisms from Aquatic Bio Systems Inc. were received with documentation stating them to be Americamysis bahia. The adult mysids used to generate the juvenile test organisms were cultured in artificial saltwater with salinity of approximately 8‰ and at a temperature of approximately 25°C. The animals were fed brine shrimp nauplii (Artemia sp. <48 hours old). Periodic lots of feed were screened for contaminants. Records of feed suppliers and the results of the contaminant analyses are on file at ABC Laboratories, Inc. A record of mysid observations and juvenile production numbers during the culture period was maintained.

Mysids were fed ad libitum brine shrimp nauplii (Artemia sp.; 24-48 hours old) at least two times daily. The concentration of the nauplii in the food stock was determined by replicate counts of aliquots pipetted onto filter paper. The food stock concentration ranged from 280 to 920 brine shrimp/mL (mean ± standard deviation [SD]: 618 ± 157 brine shrimp/mL). The food stock was enriched the day of use with a mixture of Salt Creek HUFA Enrich, AlgaMac-3000, and AlgaMac-Enhance, all of which are commercially available components. All control- and test-substance treatments received, as near as reasonably possible, the same ration (i.e., volume) of food. The enrichment mixture was prepared by diluting 1 mL of Salt Creek HUFA Enrich and 1/8 teaspoon of an approximate 40:60 AlgaMac-3000:AlgaMac Enhance mixture in a household blender with 200 mL saltwater and then blended for approximately one minute. The enrichment mixture was then added to the food stock at the rate of 50 mL/L of food stock.

The body lengths of mysids (as measured by total midline body length) were measured to the nearest 0.1 mm with a dissecting microscope.



Test type:
flow-through
Water media type:
saltwater
Limit test:
no
Total exposure duration:
28 d
Test temperature:
24.1 to 25.6°C
pH:
7.68 to 8.11
Dissolved oxygen:
5.61 to 7.97 mg/L (74 to 102% saturation)
Salinity:
7.6 to 8.4 ‰
Nominal and measured concentrations:
The testing was performed at nominal concentrations of 0 (dilution-water control), 2.0, 4.0, 8.0, 16, and 32 mg B/L.
Mean Measured Concentrations as Total Boron: 1.39 (dilution water control), 3.46, 5.58, 9.63, 18.1, and 33.0 mg B/L
Mean Measured Concentrations as Added Boron: 0.0 (dilution water control), 2.00, 4.25, 8.16, 16.6, and 31.6 mg B/L
Details on test conditions:
The dilution water was a laboratory saltwater prepared by adding a commercial sea salt mix (Crystal Sea Marine Mix; Marine Enterprises International, Inc. Baltimore, Maryland) to laboratory freshwater at a target salinity 8 ± 2‰. The laboratory freshwater consists of well water blended with well water that was demineralized by reverse osmosis to yield water with a total hardness ranging from 130 to 160 mg CaCO3/L. The dilution water was heated and aerated prior to its delivery into the diluter system. As the dilution water entered the diluter system, it passed through a particulate filter and an ultraviolet sterilizer.

During the definitive test, test solutions were delivered to three replicate test chambers and each replicate test chamber received approximately 0.67 L at each diluter cycle. The accuracy of the delivery of the test solution to the test chambers was verified by volumetric measurement before test initiation and adjusted such that the delivered volume was ±10% of the target volume.

Each test chamber used for the definitive test measured approximately 22 cm wide, 39 cm long, and 22 cm high. Each definitive-test chamber had a glass pane in the middle of the tank containing two holes near the bottom of the tank. This partition effectively cut the test chamber volume in half when the holes were stopped with silicone stoppers. The greatest depth of the test solution was approximately 12 cm, which yielded a maximum volume of approximately 10 L when one side was in use and approximately 20 L when both sides of the test chamber were in use. All test chambers were individually drained through the side of the chamber to a floor drain.

Mysids were maintained within retention baskets (constructed from nylon screening) to facilitate daily observations and enumeration. Because retention baskets were not completely isolated from one another, these retention baskets were not replicates. Only the test chamber containing the retention baskets was a true replicate.

Observations of mortality and sublethal responses were made daily for the duration of the testing period. Dead mysids were counted, differentiated by gender (if mature), and removed daily. Any missing mysids at the time of observation were noted as not found and were considered dead if not observed on following days; that mortality was subsequently reflected back to the time that the mysid was first observed missing. Any mysids that were inadvertently damaged or died as a result of becoming impinged onto the retention- or brood-basket mesh were removed from the initial number of mysids for those replicates and were not considered to be treatment-related mortality.
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
16.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
mortality
Remarks:
F0 Mysid Survival
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
growth
Remarks:
F0-Male Mysid Length
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
16.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
growth
Remarks:
F0-Female Mysid Length
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
31.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
growth
Remarks:
F0-Female Mysid Length
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
16.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
reproduction
Remarks:
mean total young per F0-female
Remarks on result:
other: added boron
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
31.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
reproduction
Remarks:
mean total young per F0-female
Remarks on result:
other: added boron
Duration:
10 d
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
mortality
Remarks:
F1 Mysid Survival
Remarks on result:
other: added boron
Reported statistics and error estimates:
Statistical analysis based on one-way ANOVA followed by Dunnett's test. ECx estimates obtained from probit method and trimmed Spearman-Karber method if poor fit in the probit approach.
Validity criteria fulfilled:
yes
Remarks:
The test acceptability criteria for this study were met. The test acceptability criteria for this study were met.
Conclusions:
The test acceptability criteria for this study were met. The water-quality characteristics remained within the tolerance limits set forth in the protocol. Survival of the dilution-water control F0 mysids was >90%. The percentage of dilution-water control F0-female mysids available to produce young that actually did produce a brood was 100%. The average total number of young produced per dilution-water control F0-female mysid was 16.0.
Based on mean total measured concentrations of boron during the 28-day exposure, the day-28 F0-mysid survival NOEC was 33.0 mg B/L, the highest boron concentration tested. The NOEC for F1-mysid survival on day 9 was 33.0 mg B/L. The 14-day F0-male mysid length was the most sensitive statistically significant endpoint and the NOEC for this parameter was 9.63 mg B/L. An EC10 could be computed for only one endpoint, mean total young per F0-female mysid, and was 16.0 mg B/L.
If the endpoints are stated based on added boron (total boron minus measured concentration natural boron in the control, Table 4), then the NOECadded born for the most sensitive endpoint was 8.16 mg Badded/L for the 14-day F0-female mysid length. The NOEC for all other endpoints, except for day-28 F0-female mysid length and mean total young per F0 female mysid, was 31.6 mg Badded/L. The NOEC for day-28 F0-female mysid length and mean total young per F0-female mysid was 16.6 mg Badded/L.
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
July 2 to 5 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Qualifier:
according to guideline
Guideline:
other: ASTM E 1440-91
Deviations:
yes
Remarks:
(Test duration extended to measure reproductive endpoints)
Principles of method if other than guideline:
The test protocol that follows is based on American Society of Testing and Materials E 1440-91 with modifications from the research publication “A 2-d Life Cycle Test with the Rotifer Brachionus calyciflorus” by Snell and Moffat (Env Toxicol Chem 11: 1249-1257 (1992)).
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The concentration of boron in test solutions was measured in composite test solution samples collected at 0 and 72 hours of the definitive test. Control and boric acid-fortified samples were also prepared for analysis at each sample period. Ten-milliliter samples were collected from the control and test substance treatment at 0 and 72 hours. Samples were diluted with reagent water to provide final sample concentrations within the analytical standard concentrations range. QC samples were prepared by fortifying laboratory saltwater with boric acid at concentrations of 5.05 and 116 mg B/L. All samples were directly analyzed using an ICP-MS system.
Vehicle:
no
Details on test solutions:
A 0.572 mg boric acid/mL primary stock solution was prepared at test initiation by weighing 0.5727 g of boric acid, adjusting for purity (99.9%) and diluting to a volume of 1,000 mL with dilution water.
Test organisms (species):
other: Brachionus calyciflorus (Rotifera)
Details on test organisms:
Brachionus calyciflorus cysts were obtained from Aquatic Eco-Systems, Apopka, Florida. Rotifer cysts were placed into Petri dishes with approximately 10 mL of warm blended freshwater. The Petri dishes were placed in an environmental chamber set at 25°C under full light for 16-20 hours. Cultures were monitored to ensure collection of larval rotifers hatched within approximately two hours of each other. Rotifers were fed an algal suspension of Nannochloropsis sp. during the test, prepared along with test concentrations at study initiation. The larvae were considered acceptable with no signs of abnormal appearance or behavior, high mortality or excessive delay in hatching. Since the culturing and testing environmental parameters were equivalent (i.e., temperature, dilution water, and lighting), no acclimation period was necessary.

Rotifers were fed an algal suspension prepared for the control and each test substance treatment. The suspension contained approximately 3 x 106 Nannochloropsis cells/mL.

Six rotifers were added to test chambers for the dilution water control and each test substance treatment. Rotifers were impartially added to a set of labeled containers with each container representing one treatment. One individual was added to each labeled container starting with the control then proceeding from the low to high test substance treatments. The individuals within each container were then transferred from each container into the corresponding test chamber using a pipet. Observations of live and dead rotifers, and rotifers with eggs were made after 72 hours.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
150 mg CaCO3/L
Test temperature:
24.6 - 24.9
pH:
7.6 - 8.2
Dissolved oxygen:
7.0 - 7.5 mg/L (89 - 94% saturation)
Nominal and measured concentrations:
Nominal Test Concentrations: 0 (control), 6.5, 13, 25, 50, and 100 mg B/L
Mean Measured Concentrations as Total Boron: 0.775 (dilution-water control), 7.13, 14.2, 25.4, 51.7, and 108 mg B/L
Mean Measured Concentrations as Added Boron: 0 (dilution-water control), 6.35, 13.4, 24.6, 51.0, and 107 mg B/L
Details on test conditions:
Brachionus calyciflorus cysts were obtained from Aquatic Eco-Systems, Apopka, Florida. Rotifer cysts were placed into Petri dishes with approximately 10 mL of warm blended freshwater. The Petri dishes were placed in an environmental chamber set at 25°C under full light for 16-20 hours. Cultures were monitored to ensure collection of larval rotifers hatched within approximately two hours of each other. Rotifers were fed an algal suspension of Nannochloropsis sp. during the test, prepared along with test concentrations at study initiation. The larvae were considered acceptable with no signs of abnormal appearance or behavior, high mortality or excessive delay in hatching. Since the culturing and testing environmental parameters were equivalent (i.e., temperature, dilution water, and lighting), no acclimation period was necessary.

The definitive and range-finding tests were conducted in 10-mL glass test tubes measuring 100 mm in height by 15 mm in diameter. The tubes contained 8 mL of control or test substance solution and were capped tightly with screw caps. The test tubes were placed in a rotating plastic bottle revolving at approximately 2 rpm. Tests were performed without light to prevent additional algal growth.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
25.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
mortality
Duration:
72 h
Dose descriptor:
LC10
Effect conc.:
26.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
mortality
Remarks on result:
other: confidence limits: 2.5 - 282.1 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
25.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
element
Remarks:
boron
Basis for effect:
other: reproduction
Details on results:
The nominal concentrations were used in the effect concentrations table, following the usual practice that a nominal concentration may be used if the measured values are reasonably close to the nominal.
Observations of live and dead rotifers, and rotifers with eggs were made after 72 hours. After 72 hours, the mean number of surviving rotifers was 21.2, 14.2, 21.4, 20.2, 7.2, and 5.0 live rotifers in the 0 (control), 6.5, 13, 25, 50, and 100 mg B/L treatments, respectively. One dead rotifer was observed in the 6.5, 13 and 50 mg B/L test substance treatments and four dead rotifers were observed in the 25 and 100 mg B/L treatments. Rotifers with eggs were observed in all replicates of all test substance treatments except replicates A and B of the 6.5 mg B/L treatment. After 72 hours, the mean number of rotifer reproduction was 15.2, 8.2, 15.4, 17.3, 2.0, and 0 rotifers in the 0 (control), 6.5, 13, 25, 50, and 100 mg B/L treatments, respectively.

NOEC and LC10 values have been calculated for the endpoint mortality. Preference is given to the use of the LC10 (26.6 mg/L) compared to the NOEC (25.4 mg/L ) value for mortality. For the endpoint reproduction, both the NOEC (25.4 mg/L) and EC10 (39.9 mg/L) values could be calculated. However the EC10 for the endpoint reproduction was not reliable because of the large confidence interval between 0 mg/L - infinity.
Reported statistics and error estimates:
All statistical analyses were performed using SAS software (SAS version 9.1 for Windows). Inferences of statistical significance were based upon a p = 0.05 unless otherwise noted.
The no-observed-effect concentration (NOEC) and lowest-observed-effect concentration (LOEC) for mortality/immobility data were determined by using a one-tailed Dunnett’s test.
The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A
Validity criteria fulfilled:
yes
Conclusions:
The nominal NOEC and LOEC for both survival and reproduction were 25 and 50 mg B/L, respectively.Based on mean measured total boron concentrations, the 72-hour EC50 for survival and reproduction were estimated to be 48.3 mg B/L and 50.0 mg B/L, respectivly (95% confidence limits could not be determined). The slope of the 72-hour concentration-response could not be calculated.
Based on the added measured concentrations the NOEC and LOEC for survival and reproduction were 24.6 and 51.0 mg B/L, respectively.
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions. Peer-reviewed technical publication. Study meets guidelines except that temperature was elevated to 24 °C to accelerate growth and test duration was shorter (14d) than current methods.
Principles of method if other than guideline:
Two replicate 14-day static renewal chronic toxicity tests were conducted with Daphnia magna Straus
GLP compliance:
not specified
Analytical monitoring:
yes
Vehicle:
no
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain/clone: Straus
- Source: Laboratory reared
- Feeding during test
- Food type: Selenastrum capricornutum
- Amount: 2.5 mg dry wt./L of water
- Frequency: 3 days / week
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
14 d
Hardness:
170 mg/L as CaCO3
Test temperature:
23 to 25.2°C
pH:
7.3 - 8.2
Dissolved oxygen:
> 90% saturation (range 8.3 to 8.8 mg/L)
Nominal and measured concentrations:
all analyzed concentrations were within a range of 98.5 and 111.4 % of nominal.
Measured: 7.4, 13.8, 28.1, 57.4, 107.4 mg/L in Test I; 7.8, 14.3, 28.9, 60.0, 113.2 mg/L in Test II
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 600 mL glass beaker, each containing five glass tubes (2.5 x 12.5 cm) with 363 µm mesh bottoms. The tubes were supported about 1.0 cm off of the bottom of the beaker with a 1.0 mm mesh stainless steel platform. During the studies, each beaker contained the appropriate amount of food, dilution water and test material made up to a 500 mL volume.
- Aeration: gentle
- Renewal rate of test solution (frequency/flow rate): 3 times / week
- No. of organisms per vessel: One neonate daphnid in each uniquely labeled tube.
- No. of vessels per concentration (replicates): 4x5 = 20



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Lake Huron water. This water was obtained from the Midland Water Treatment Plant prior to final chlorination and was adjusted to a hardness of about 170 mg/L (as CaCO3) prior to autoclaving. The water was autoclaved at 121 °C and 124 kPa for 35 minutes.
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH, D.O., and temperature were also measured and recorded on each renewal day.


OTHER TEST CONDITIONS
- Photoperiod: 16 hr light/8 hr dark
- Light intensity: +- 1700 - 2400 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The reproductive endpoint was obtained by hand counting the neonates on a Monday, Wednesday, and Friday basis. Growth was calculated by determining the dry weight of the surviving adult organisms at the tests end. Survival data were collected every Monday, Wednesday, and Friday
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
13.8 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
mortality
Remarks on result:
other: Test I
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
13.8 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
reproduction
Remarks on result:
other: Test I
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
13.8 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
growth
Remarks on result:
other: Test I
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
14.3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
mortality
Remarks on result:
other: Test II
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
14.3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
reproduction
Remarks on result:
other: Test II
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
14.3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
growth
Remarks on result:
other: Test II
Duration:
14 d
Dose descriptor:
LC10
Effect conc.:
24 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
mortality
Remarks on result:
other: Test I; confidence limits: 17.2 - 33.8 mg/L
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
16.6 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
reproduction
Remarks on result:
other: Test I; confidence limits: 13.7 - 18.9 mg/L
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
20.6 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
growth
Remarks on result:
other: Test I; confidence limits: 0.2 - 2002 mg/L
Duration:
14 d
Dose descriptor:
LC10
Effect conc.:
25.8 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
mortality
Remarks on result:
other: Test II; confidence limits: 24.6 - 26.3 mg/L
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
16.6 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
reproduction
Remarks on result:
other: Test II; confidence limits: 15.5 - 17.2 mg/L
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
13.2 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
element
Basis for effect:
growth
Remarks on result:
other: Test II; confidence limits: 13.0 - 13.7 mg/L
Details on results:
NOEC and LC10/EC10 values are provided for the endpoints reported, i.e. mortality, reproduction and growth. Preference is given to the use of EC10/LC10 values for PNEC derivation
Reported statistics and error estimates:
Data derived from the studies were analyzed by a one-tailed Dunnett's test (alpha = 0.05). The Dunnett's procedure used simultaneously tested for heterogeneity of variances using Bartlett's test. If the variances were heterogeneous the Wilcoxon signed rank test was used to compare the means (Hollander and Wolfe 1973). Mean comparisons between test and control concentrations were performed on the following endpoints: percent survival, mean total young/adult, mean brood size/adult and mean dry weight/adult.

The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A.
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well performed and documented study according to GLP and international guidelines
Qualifier:
according to guideline
Guideline:
other: ASTM E 2455-6 (2006). Standard guide for conducting laboratory toxicity tests with freshwater mussels.
Principles of method if other than guideline:
Test methods followed those outlined in the previously approved Study Protocol Water-only Chronic Toxicity Testing of Boron with the Freshwater Fatmucket Mussel (Lampsilis siliquoidea). Amendments and deviations are documented in Attachment 1. The Study Protocol was also based on ASTM E2455-06 (ASTM, 2006).
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Test organisms (species):
other aquatic mollusc: Lampsilis siliquoidea
Details on test organisms:
- Source: Missouri state university (MSU).
- Gravid female L. siliquoidea were collected in Missouri and glochidia obtained were inoculated onto largemouth bass (Micropterus salmoides) at the MSU propagation facility. Juvenile mussel drop-off occured approx 3 weeks after inoculation. The drop-off date for the batch of mussels used for testing was Dec 1 to 5, 2010.
- Juvenile L.siliquoidea were reared at MSU in semi-closed systems for approx. 2 months at temperature between 20 and 25 °C in St Jopseph River Water.
- Feeding was based on recommendations of the ASTM guidelines.
- Juvenile mussels were received at Envrion for acclimation on Feb 8, 2011 and maintained in clean control water until commencement of dosing on Feb 17, 2011.
- Feeding consisted of a suspenseion of Nannochloropsis algae and Reed mariculture "shellfish diet". A stock solution was prepared from 100 ml of culture water by adding 0.42 ml of Nannochloripsis concentrate and 1.4 ml of shellfish diet concentrate. THe stock solution was fed at the rat of 1 ml/L holding water.
Test type:
semi-static
Water media type:
freshwater
Total exposure duration:
21 d
Test temperature:
18.9-21.7 °C
pH:
6.82 to 7.93 s.u.
Dissolved oxygen:
6.5 to 10.5 mg/L
Nominal and measured concentrations:
0 (control), 10.0, 17.8, 31.6, 56.1 and 100 mg B/L
(Average measured boron concentrations were within 3 percent of nominal values; maximum deviation from nominal values was 12 percent)
Details on test conditions:
TEST SYSTEM
- 8 individual replicates for determination of test endpoints
- Each replicate contained 5 juvenile L.siliquoidea
- Test vessels consisted of 60 ml non-eactive plastic vessels, each containing 40 ml of control or test solution

TEST MEDIUM / WATER PARAMETERS
- hardness: 88.8 to 108 mg CaCO3/L
- alkalinity: 45 to 50 mg C03/L
- total ammonia: < 0.1 to 0.13 mg/L as N

OTHER TEST CONDITIONS
- photoperiod: 16hr light, 6hr dark
- light intensity: 233-398 lux
- feeding: mussel feeding was accomplished by ading dilute food suspension daily to renewal waters prior to daily water renewal. At day 5 of the study, test organisms feeding was reduced by 2.3 due to the build-up of mussel pseudofeces in the test vessels. this deviation of the protocol has been documented.
- test solution: test solution were prepared for initiating dosing and weekly thereafter until test termination. Ten liters of control water were placed in separate 10 L collapsible plastic cubitainers® and boric acid was added directly to the cubitainers® to prepare the appropriate boron concentration. The cubitainers® were capped and shaken by hand until all boron was dissolved, and solutions were not used for approximately 24 hours before dosing. Solutions were refrigerated at 4 C when not in use. Each batch of water was used for seven daily renewal events.
- At test termination (day 21), 2 to 3 drops of a sugar/formalin solution were added to the test vessels to immobilize the mussels. Any observations of general differences in size, condition, etc. of test organisms in different boron exposures were also noted at this time. Immobilized surviving mussels were measured under 50X magnification to the nearest 0.01 mm.

CONTROL AND DILUTION WATER
- Reconstituted moderately hard laboratory water (US EPA, 2002) served as the control water in all test and control exposures.
- Control and test exposures were renewed daily (at 80 to 90 percent of the total replicate solution volume) from stocks prepared at the beginning of each week.

RANGE FINDING TEST
A semi flow-through 28 d range-finding test was conducted from November 19 to December 17, 2010, and utilized seven two-month old L. siliquoidea in each of two replicate natural whole sediment exposures. Natural sediment (West Bearskin Lake, Cook County, MN) was obtained from LimnoLogic Inc., Duluth, MN (collected October 29, 2010). Nominal whole sediment boron concentrations ranged from 208 to 3,333 mg B/Kg sediment dry weight (25 to 400 mg B/kg sediment wet weight) and were based on unpublished data for a range of aquatic taxa. These nominal wet weight sediment boron concentrations (25 to 400 mg B/kg sediment) provided data useful in selection of the water concentrations utilized in the present study (i.e., 10 to 100 mg B/L). Selected water quality parameters were evaluated to document that adequate overlying water quality conditions were maintained under the semi flow-through test format. Boron concentrations in the sediment and the overlying water were monitored to determine whether boron concentrations in the sediment were stable and approximated nominal concentrations. There was no significant mortality of mussels in any exposure. Overlying water, whole sediment, and sediment extract water were measured from these replicates to verify boron movement from sediment to water for design of the definitive toxicity test.
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
mortality
Remarks on result:
other: analytical confirmation of B concentrations
Duration:
21 d
Dose descriptor:
LC10
Effect conc.:
40.4 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
mortality
Remarks on result:
other: analytical confirmation of B concentrations; confidence limits: 21.9 - 74.6 mg/L
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: biomass shell length
Remarks on result:
other: analytical confirmation of B concentrations
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
30 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: biomass shell length
Remarks on result:
other: analytical confirmation of B concentrations; confidence limits: 21.7 - 41.5 mg/L
Reported statistics and error estimates:
The 21 day survival and biomass data were assessed with the ToxCalcTM v 5.0.23 (TIDEPOOL Scientific Software). Both point estimate endpoints (EC25 and IC25 or IC50 values) and statistical hypothesis (NOEC and LOEC) values were calculated for the total number of mussels (i.e., survival) and biomass (shell length) data. Assessments of statistical significance in NOEC and LOEC determinations were conducted at the 95 percent confidence level (alpha 0.05).
The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A
Conclusions:
Based on survival, the IC25 and IC50 values (and associated 95 percent CIs) were 38.4 (37.1 – 39.5) and 46.5 (44.8 – 48.4) mg B/L, respectively. The test endpoints were based on nominal boron concentrations because all measured boron exposure concentrations were within 12 percent of nominal values, and average measured boron concentrations were within three percent of nominal values. The NOEC and LOEC values (31.6 and 56.1 mg B/L, respectively) bracketed the IC25 value, indicating concurrence of statistical measurements.

Based on the average biomass (shell length) of surviving mussels, the IC25 and IC50 values (and associated 95 percent CIs) were 34.6 (32.8 – 36.1) and 44.4 (42.5 – 46.9) mg B/L, respectively. The shell length NOEC and LOEC values (10.0 and 17.8 mg B/L, respectively) were below the IC25 value due to the high test precision associated with these data. Thus, the IC25 value may best reflect the threshold toxicity concentration for Lampsilis siliquoidea in water-only test exposures.
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study, pre OECD guidline
Qualifier:
no guideline followed
Principles of method if other than guideline:
A chronic semi-static procedure was followed in conducting the 21 day chronic toxicity test.
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
Water samples were collected for analytical verfification of boron from one test chamber in the control and in each of the test concentrations. One set of water samples was collected from the "old" test solutions and one set was collected also from the "new" test solutions at the time of water renewal.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Source: obtained from a stock culture
- Feeding during test : The daily diet ocnsisted of 0.15 ml of a 5:1 mixture of Ralston Purina Trout Chow and dehydrated alfalfa, both certified pesticide-free, in deionized water. The diet was supplemented with 1 ml of an alga suspension containing approximately 20 million cells of the green alga, Selenastrum capricornutum Printz, per ml of AAP medium (U.S. EPA 1971)


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
On the day preceding test initiation, reproductively mature adults were isolated. The young produced by these adults were removed and used in a test within 24h.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
166 (range 135 - 217) mg/L as CaCO3
Test temperature:
19.2 (range 18-21) °C
pH:
7.1 - 8.7
Dissolved oxygen:
> 9 mg/l ( > 90% saturation)
Nominal and measured concentrations:
The measured boron concentrations in the test waters exceeded 95% of the corresponding nominal values.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: 250 ml glass beakers containing 200 ml of the test solution
- Aeration:
- Renewal rate of test solution (frequency/flow rate): 3 times per week
- No. of organisms per vessel: 7 contained one daphnid and 3 contained 5 daphnid
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 20 ( 6 containing five daphnids and 14 containing one daphnid)


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Carbon filtered, well water which has been described previously (Maki and Bishop 1979)


OTHER TEST CONDITIONS
- Photoperiod: 16h light : 8h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Adult survival and number of young were recorded daily for each test chamber. Prior to renewal the young produced by adults in the three beakers containing five organisms were discared. Surviving adults in these test chambers were enumerated and retained. Surviving adults and juveniles in the remaining seven beakers were enumerated after which juveniles and dead adults were discarded.
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
18.2 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
reproduction
Remarks on result:
other: analytical confirmation; confidence limits: 2.8 - 120.1 mg/L
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
27 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
growth
Remarks on result:
other: analytical confirmation
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
6 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
reproduction
Remarks on result:
other: analytical confirmation
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
52.9 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
growth
Remarks on result:
other: analytical confirmation; confidence limits: 49.6 - 56.3 mg/L
Details on results:
NOEC and EC10 values are provided for the endpoints reported, i.e. reproduction and growth. Preference is given to the use of EC10 values for PNEC derivation
Reported statistics and error estimates:
The LC10/EC10 values have been recalculated with the log-logistic model using Toxicity Relationship Analysis Program, Version 1.21A. T-tests were used to analyze differences in mean brood sizes and in daphnid length. Length of the adults surviving 21 days of exposure were determined from the apex of the helmet to the base of the spine. All statistical procedures followed ZAR (1974)

Description of key information

Long-term effects (LC10/NOEC) on reproduction on freshwater vertebrates ranged from 6.6 to 32 mg B/L based on several well-accepted guideline studies.

Key value for chemical safety assessment

Additional information