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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:
key study
Study period:
1999-04-30 - 1999-07-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to
Guideline:
other: OECD 202 / part II
Deviations:
no
GLP compliance:
yes
Remarks:
GLP - Rule DINQP-093-INMETRO-Nov/98
Specific details on test material used for the study:
One primary stock solution was prepared by weighing 10 mg of Ferbam dissolving with 2 mL of acetone then this stock solution was added reconstituted water to a total volume of 100 mL and it was stirred by 5 minutes (this stock solution was used by determined 48-hours EC50 - Acute Immobilization Test). Second stock solution was prepared by weighing 10 mg of Ferbam dissolving with 2 mL of acetone then this stock solution was added reconstituted water to a total volume of 100 mL and it was stirred by 5 minutes (this stock solution was used in the definitive test -Chronic test). Lower test concentrations were prepared by dilution of second stock solution and were listed in raw data.
Analytical monitoring:
not specified
Details on sampling:
Surviving Daphnia magna and their progeny were counted and recorded on days 0, 2, 5, 7, 9, 12, 14, 16, 19 and 21.
Each new test concentration and control were analysed for alkalinity, conconductivity, hardness, pH, dissolved oxigen on test day 0, 2, 5, 7, 9, 12, 14, 16, 19 and 21. Each descarted test concentration and control were analysed pH and dissolved oxigen.
Vehicle:
not specified
Details on test solutions:
Test Concentration
48 hours-EC50 obtained of the Acute Immobilization Test was used to determine the concentration levels in the Chronic Test.
The range-finding (Acute Immobilization Test) was carried out with 0.032; 0.056; 0.10; 0.32 and 0.56 mg/L.
Based in the 48 hours-EC50 of 0.097 mg/L the chronic test was carried out with eight test concentrations 0.10; 0.055; 0.03; 0.016; 0.009; 0.005; 0.0028 and 0.0015 mg/L.
Test organisms (species):
Daphnia magna
Details on test organisms:
Source of organism
The test organism used was microcrustaceous Daphnia magna obtained from the Carolina Biological Supply Company, Burlington, North Carolina, and maintained in stock culture at BIOAGRI.
Stock culture
This organisms are maintained in stock culture under reconstituted natural water with adjusted pH to 7.4 + 0.2 and controlled environmental conditions such as temperature of 20±2°C and photoperiod of 16 of light and 8 hours of darkness.
First instar daphnids < 24 hours old were used to start the test. All organisms used were originated from the same source and same culture container.
Acclimation
Twenty four hours prior to test adults with full brood chambers were isolated into test water. The following morning the newly released instars, less than 20 hours old, were carefully removed with a wide bore pipette and fed for one hour prior to initiation of the chronic test.
Feeding
During the test, daphnids were fed with algae and with the same frequency as that used for culturing in the brood stock.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
242 mg/L (water parameter at 0-hour measured)
Test temperature:
20 +- 2 °C (water parameter at 0-hour measured)
pH:
7.68 (water parameter at 0-hour measured)
Dissolved oxygen:
5.3 mg/L (water parameter at 0-hour measured)
Salinity:
not applicable
Conductivity:
378 uS/cm (water parameter at 0-hour measured)
Nominal and measured concentrations:
nominal test concentrations:
0.032 mg/L
0.056 mg/L
0.1 mg/L
0.32 mg/L
0.56 mg/L
Details on test conditions:
Test System
The range-finding test was carried out in static system without renewal of test concentration for 48 hours.
Chronic test was carried out in a semi-static system, with renewal of test concentration during the exposition. The solutions were renewed on each Monday, Wednesday and Friday.

Test containers
It was used 40 mL of test concentration for each organism. It was used 250 mL -glass beaker containing 200 mL of test concentration for the chronic test.
Number of organisms
It was used 40 organisms divided into eight groups of five organisms each for each test concentration and for the control.

Light and Temperature
A light-dark cycle was used: 8 hours darkness and 16 hours light. The temperature was maintained 20 +/- 2°C.

Reference test
The reference test using potassium dichromate (Merck Batch 1048640500; expired date 31/january/2003) was conducted in order to verify the sensibility of test- organism. It was carried out as mentioned for the range finding, but using four replicates of following concentrations: 0.10; 0.32; 0.56 and 1 mg/L.

Glassware preparation
All glassware prior the test, were washed with commercial detergent solution. After rinsed with tap water, they were washed with commercial acetone, sulphocromic solution, and then nitric acid 5% (v/v) to remove any residue. They were washed again in tap water followed by rinsing with distilled and deionized water.

Statistical Analysis Method
Analysis of variance were used to test for significant test substance effects . The statistical tests employed and the results of these tests should be reported in raw data.
Data were calculated as means, standard deviations and confidence limits for control and exposure replicates measurements. The means were compared with the control using the Dunnett'Test (95% confidence limits) to calculate the no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) for daphnids reproduction. And for lethal effect were used the DUNCAN TEST (95% confidence limits), which compared the lethal effect of the control with replication. The computer program used was Toxstat 3.3. (GULLEY et al., 1991).
Based on the data obtained from the reference test, the EC50 and 95% confidence limits were determined using the Trimmed Spearman- Karber Method (HAMILTON et al.,1978).
Reference substance (positive control):
yes
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.018 mg/L
Nominal / measured:
not specified
Conc. based on:
other: tested concentrations
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.003 mg/L
Nominal / measured:
not specified
Conc. based on:
other: tested concentrations
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
other: MATC
Effect conc.:
0.004 mg/L
Nominal / measured:
not specified
Conc. based on:
other: tested concentrations
Basis for effect:
immobilisation
Details on results:
Results of Acute Test
The 48 hours-EC50 values of the range-finding test was 0.097 mg/L (95% confidence limits = 0.08 -0.12 mg/L).
The lethal effects were showed in Table 2.
There was a significant toxic effect on the adult gereration resulting in 100% mortality in the 0.030; 0.055 and 0.10 mg/L by day 5.
There was a sub-lethal effect for 0.016 mg/L as the total number of young produced was approximately 35% of the total number produced by the control as a result of the initial acute toxicity response. This group showed 35%» of immobilization by day 21.

Reproduction
There was significant (T test p=0.05) difference between test concentration 0.009 and 0.005 mg/L and water control byr day 21. There was no significant (T test p=0.05) differrence between test concentration 0.0028 and 0.0015 mg/L and water control by day 21.
By the 8th day Daphnia water control had released their first brood.

No Observed Effect Concentration
At test termination the average cumulative daphnid young produced of the eight replicates and immobilization results showed were not significantly different (p=0.05) to 0.0028 mg/L (NOEC).
At test termination the average cumulative daphnid young produced of the eight replicates and immobilization results showed were significantly different (p=0.05) to 0.005 mg/L.

The MATC was 0.0037 mg/L.
Results with reference substance (positive control):
The 48 hours - EC50 values based on adult immobility with corresponding 95% confidence limits for the reference compound, potassium dichromate, was 0.56 mg/L (0.50 -0.64 mg/L), respectively.
Table 1: Chemical Analytical Determinations
Nominal Concentration*
mg/L
0
(05/05)
2
(07/05)
5
(10/05)
7
(12/05)
9
(14/05)
12
(17/05)
14
(19/05)
16
(21/05)
19
(24/05)
Average Effective Conc.
(mg/L)
100 100.01 100.03 100.2 99.874 100.10 100.31 100.12 100.17 89.139 98.893
1 0.907 1.01 1.01 0.997 1.01 1.01 1.01 1.01 0.89 0.984
0.1 0.140 0.102 0.110 0.101 0.110 0.10 0.10 0.103 0.09 0.106
Control 0 0 0 0 0 0 0 0 0 0
* Stock Solution

Table 2: EC50 values (mg/L)
Time EC50* 95% confídence limits
Days 5-14 0.0219 mg/L Are not realiable
Day 16 0.0194 mg/L 0.02-0.02
Day 19 0.0185 mg/L 0.02-0.02
Day 21 0.0180 mg/L 0.02-0.02
* Spearman-Karber Method

Table 3: Cumulative Immobilization by 21 days
Nominal concentration mg/L Cumulative Immobilization
0,1 40*
0,055 40*
0,03 40*
0,16 13*
0,009 0
0,005 0
0,0028 0
0,0015 0
Solvent 0
Control 0
Control 0
Data following the same signal are signifaicantly different (p=0.05), Fisher's test

Table 4: Average Cumulative Daphnid young produced by 21 days
Nominal concentration mg/L Average Cumulative production of the eight replicates a
0.009 538.25*
0.005 584.50*
0.0028 658.37
0.0015 671.37
Solvent
Control
659.00
Control 673.25
a Repesentthe average production of the eight replicates.
Data following the same signal are significantly different (p=0.05, Dunnett's Test.
Validity criteria fulfilled:
yes
Conclusions:
The 48 hours-EC50 values of the range-finding test was 0.097 mg/L (95% confidence limits = 0.08 -0.12 mg/L).
The 48 hours - EC50 values based on adult immobility with corresponding 95% confidence limits for the reference compound, potassium dichromate, was 0.56 mg/L (0.50 -0.64 mg/L), respectively.
At test termination the average cumulative daphnid young produced of the eight replicates and immobilization results showed were not significantly different (p=0.05) to 0.0028 mg/L (NOEC).
At test termination the average cumulative daphnid young produced of the eight replicates and immobilization results showed were significantly different (p=0.05) to 0.005 mg/L.
The MATC was 0.0037 mg/L.
Executive summary:

A 21-day chronic toxicity study of Ferbam to Daphnia magna was conducted according to GLP and OECD 202 / part II. The range-finding test was carried out in static system without renewal of test concentration for 48 hours. Twenty young daphnids (maximum 24 h-old) were divided into four groups of five organisms each, it were used at each test concentration and for controls. The nominal test concentration were: 0.032; 0.056; 0.1; 0.32 and 0.56 mg/L. The test flasks were incubated with light-dark cycle of 8 hours darkness and 16 hours light and the temperature was maintained 20 + 2°C during 48 hours. Based on EC50-48 hours of 0.097 mg/L the definitive chronic test was carried out in semi-static system with fourty young daphnids (maximum 24 h-old) divided into eight groups of five organisms each, it were used at each test concentration and for the control. The nominal test concentration were controls; 0.0015; 0.0028; 0.005; 0.0009; 0.016; 0.03; 0.055 and 0.10 mg/L for evaluation survival and reproduction. Water quality parameters such as dissolved oxygen, conductivity, pH, hardness and alkalinity were measured throughout the test and were within acceptable limits.

From the data collected during this study, a 21-days EC50 was calculated to be 0.0180 mg/L (95% confidence limits = 0.02 - 0.02 mg/L) based on the tested concentration. At test termination the average cumulative daphnid young produced of the eight replicates and immobilization results showed were not significantly different (p=0.05) to 0.0028 mg/L (NOEC). At test termination the average cumulative daphnid young produced of the eight replicates and immobilization results showed were significantly different (p=0.05) to 0.005 mg/L. The MATC was 0.0037 mg/L. By the 8th day Daphnia at all exposure concentration had released their first brood in the water control.

As a quality check the daphnids were challenged with a reference compound, potassium dichromate, which 48-hours EC50 value was 0.56 mg/L.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-02-24 - 2009-06-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPPTS 850.1350 (Mysid Chronic Toxicity Test)
Version / remarks:
1996
Deviations:
no
Qualifier:
according to
Guideline:
other: ASTM Standard E1191-03a
Version / remarks:
Standard Guide for Conducting Life-Cycle Toxicity Tests with Saltwater Mysids
Deviations:
no
GLP compliance:
yes
Remarks:
U.S. EPA (40 CFR Parts 160 and 792, 17 August 1989, OECD (ENV/MC/Chem (98) 17), Japan MAFF (11 NohSan, Notification No. 6283, Agricultural Production Bureau, 1 October 1999)
Analytical monitoring:
not specified
Details on sampling:
Water samples were collected from one test chamber of each treatment and control group one day prior to the start of the test after conditioning the diluter for approximately five hours. Water samples also were collected from alternating replicate test chambers in each treatment and control group at the beginning of the test, at approximately weekly intervals during the test and at test termination to measure concentrations of the test substance. The samples were collected from mid-depth, placed in glass vials, and processed immediately for analysis.
Vehicle:
not specified
Details on test solutions:
Dilution water
The water used for culturing and testing was natural seawater collected at Indian River Inlet, Delaware. The freshly-collected seawater was passed through a sand filter to remove particles greater than approximately 25 μm, and pumped into a 37,800-L storage tank. The filtered saltwater then was diluted to a salinity of approximately 20‰ with freshwater from a well on the Wildlife International, Ltd. site and was aerated with spray nozzles. Prior to use, the 20‰ water was filtered to 0.45 μm to remove
fine particles and was passed through an ultraviolet (UV) sterilizer.
Test organisms (species):
Americamysis bahia (previous name: Mysidopsis bahia)
Details on test organisms:
The saltwater mysid (Americamysis bahia) was selected as the test species for this study. The saltwater mysid is representative of an important group of marine invertebrates and was selected for use in the test based upon past use in the laboratory. Mysids used in the test were obtained as juveniles (<24 hours old) from cultures maintained by Wildlife International, Ltd., Easton, Maryland.
Adult mysids in the cultures were held in the laboratory for at least 14 days before juveniles were collected for testing. Cultures were maintained in a recirculating saltwater system kept at approximately 27°C. Saltwater was filtered through a biofilter and UV sterilized prior to entering the recirculating system. During the two week period immediately preceding the test, water temperatures in the cultures ranged from 24.8 to 27.6ºC, measured with a liquid-in-glass thermometer. The pH of the water ranged from 7.9 to 8.1, measured with a Fisher Scientific Accumet Model 915 pH meter. Dissolved oxygen ranged from 6.5 to 7.5 mg/L (≥89% of saturation), measured with a Yellow Springs Instruments Model 85 dissolved oxygen meter. Salinity of the filtered saltwater ranged from 20 to 21 parts per thousand (‰), measured with a Vitalsine refractometer.
Mysids in the cultures were fed live brine shrimp nauplii (Artemia sp.) daily, supplied by Brine Shrimp Direct, Ogden, Utah. During the test, the mysids were fed live brine shrimp nauplii (Artemia sp.) up to four times daily. To initiate the test, the juvenile mysids (<24 hours old) were collected from the cultures and impartially distributed one and two at a time into transfer containers until each container held 15 mysids. Each group of mysids then was transferred to an indiscriminately assigned test compartment. All
transfers were performed using wide-bore pipettes below the water surface.
Test type:
flow-through
Water media type:
saltwater
Limit test:
no
Total exposure duration:
28 d
Hardness:
not specifed
Test temperature:
25 +/- 2 °C
pH:
7.9 - 8.1
Dissolved oxygen:
6.7 +/- 1.13 mg/L
Salinity:
20 %o
Conductivity:
not specified
Nominal and measured concentrations:
nominal
0.013 mg/mL
0.025 mg/mL
0.050 mg/mL
0.10 mg/mL
0.20 mg/mL

measured (mean)
0.012 mg/mL
0.023 mg/mL
0.044 mg/mL
0.091 mg/mL
0.18 mg/mL
Details on test conditions:
Test Apparatus
The toxicity test was conducted using an exposure system consisting of a continuous-flow diluter used to deliver each concentration of the test substance, a solvent control and a negative control (dilution water) to test chambers. Sixty milliliter syringes containing the concentration-specific stock solution (see Preparation of Test Concentrations) were placed on syringe pumps for delivery of the stock solutions to the mixing chambers. Syringe pumps (Harvard Apparatus, Massachusetts) delivered the test substancestock solutions or solvent to impartially assigned mixing chambers where the stocks or solvent were mixed with dilution water prior to delivery to the test chambers. The flow of dilution water into each mixing chamber was controlled using rotameters, and was adjusted to provide each juvenile test chamber with at least 18 volume additions of test water per day and adult test chambers with at least 6 volume additions of test water per day. After mixing, the flow from each mixing chamber was split to deliver test water to four replicate test chambers.
The syringe pumps used to deliver stock solutions or solvent to the mixing chambers were calibrated prior to the test. The rotameters used to control the flow of dilution water to the mixing chambers were calibrated prior to the test and verified or recalibrated approximately weekly during the test. The proportion of the test water that was split into each replicate test chamber was checked prior to the test and approximately weekly during the test to ensure that flow rates varied by no more than ± 10% of the mean flow rate for the four replicates. Delivery of test solutions to the test chambers was initiated one day prior to the introduction of the test organisms to the test water in order to achieve equilibrium of the test substance. The general operation of the exposure system was checked visually at least once on the first and last days of the test and at least two times per day during the test.
The delivery system and the test chambers were placed in a temperature-controlled environmental chamber to maintain the target temperature throughout the test period. All test chambers were labeled with the project number, test concentration and replicate designation. Prior to pairing, mysids in each treatment and control group were held in one test compartment placed in each of four replicate test chambers. The compartments were approximately 2-L glass containers measuring approximately 12 cm in diameter and 19 cm in height, with two nylon mesh covered holes on opposite sides of the container. The compartments were placed in 9-L glass aquaria containing approximately 2 to 3 L of test solution. The depth of water in a representative test chamber and a test compartment was 8.0 and 7.4 cm, respectively.
After mysids attained sexual maturity and were paired on Day 15, reproductive pairs were placed in reproductive compartments, one pair per compartment, with up to five paired compartments in each replicate test chamber. The reproductive compartments were approximately 10-cm diameter glass petri dishes with sides of nylon mesh screen. The reproductive compartments were placed in 19-L glass aquaria filled with approximately 14.5-L of test solution, which contained a self-starting siphoning system to help facilitate the exchange of test solution. The depth of water in a representative test chamber and reproductive compartment was 14.5 and 13.2 cm, respectively.

Preparation of Test Concentrations
One stock solution of radiolabelled Ferbam was prepared in dimethylformamide (DMF) at a nominal concentration of approximately 100 mg/L. The radioactivity of the stocks was confirmed prior to use in the study by analyzing triplicate 20-μL samples of the stock solution by liquid scintillation counting (LSC). The analysis resulted in a mean measured concentration of 122 mg/L corresponding to 122% of the expected radioactivity. A primary stock solution of non-radiolabelled Ferbam was prepared for the study by dissolving non-radiolabelled Ferbam in DMF at a nominal concentration of 1.0 mg/mL.
Dispensing stock solutions at nominal concentrations of 0.013, 0.025, 0.050, 0.10 and 0.20 mg/mL were prepared in DMF using calculated volumes of the radiolabelled and non-radiolabelled stock solutions to achieve a nominal radioactivity of 5 x 105 dpm/mL in each dispensing stock. The stock solutions were mixed by inversion, and ranged in appearance from clear and light brown to clear and dark brown. Stock solutions were stored refrigerated in glass amber bottles with Teflon®-lined lids Every 1 to 3 days during the study, the syringes were purged of any old stock and refilled with aliquots of each stock.
Prior to pairing of the mysids, the five test substance stock solutions were injected into the diluter mixing chambers at a rate of 12.50 μL/minute where they were mixed with dilution water delivered at a rate of 125 mL/minute to achieve the desired test concentrations. Following pairing, the stock solutions were injected into the diluter mixing chambers at a rate of 25.0 μL/minute where they were mixed with dilution water delivered at a rate of 250 mL/minute to achieve the desired test concentrations and nominal radioactivity of 50 dpm/mL (Appendix 4.3). The toxicant and dilution water flows were terminated in the 20 μg/L test concentration following pairing due to low juvenile survival at this concentration. The negative control received dilution water only. The solvent control was prepared by delivering HPLCgrade DMF to the mixing chamber for the solvent control. The concentration of DMF in the solvent control and all 14C-Ferbam treatment groups was 0.1 mL/L.

Environmental Conditions
Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight were controlled by an automatic timer to provide a photoperiod of 14 hours of light and 10 hours of darkness. A 120-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in lighting. Light intensity was measured at the water surface of one representative test chamber at test initiation using a SPER
Scientific Model 840006C light meter.
The target test temperature during the study was 25 ± 2°C. Temperature was measured in each test chamber at the beginning and end of the test, and weekly during the test, using a hand-held liquid-in glass thermometer. Temperature also was monitored continuously in one negative control test chamber using a Fulscope ER/C Recorder, which was verified prior to test initiation and verified or calibrated approximately weekly during the test with a hand-held liquid-in-glass thermometer.
Prior to pairing, dissolved oxygen was measured in one replicate test chamber of each treatment and control group at the beginning of the test and at approximately weekly intervals during the test period, with measurements typically rotating among the replicates in each group at each measurement interval. After mysids attained sexual maturity and were paired on Day 15, gentle aeration was added to each test chamber, and dissolved oxygen was measured daily until the end of the test in one replicate test chamber of each treatment and control group, with measurements typically rotating among the replicates in each group at each measurement interval. Dissolved oxygen was measured using a Thermo Orion Model 850Aplus dissolved oxygen meter.
Measurements of pH were made in one replicate test chamber of each treatment and control group at the beginning and end of the test, and at approximately weekly intervals during the test, with measurements typically rotating among the replicates in each group at each measurement interval. Measurements of pH were made using a Thermo Orion Model 525Aplus meter. Salinity was measured daily in one replicate of the negative control, with measurements typically rotating among the replicates
in the group at each measurement interval. Salinity was measured using a Vitalsine refractometer. If 100% mortality occurred in a test chamber, measurements of temperature, dissolved oxygen and pH were taken in that test chamber and then discontinued.

Biological Observations and Measurements
Observations of the survival and behavior of each first-generation mysid were made daily throughout the test. The criteria for death included lack of movement, absence of respiratory movements, and lack of reaction to gentle prodding. At pairing on Day 15, the sex and maturity of each mysid was determined by microscopic examination, and, when possible, five male/female pairs were assigned to reproductive compartments in each replicate test chamber, with one pair per compartment. Any immature
mysids or extra females were discarded at this time. Any sexually mature males remaining after pairing were maintained in a separate compartment within the respective replicate test chamber. If a male in a reproductive compartment died, it was replaced with a male from the pool of males maintained in the same replicate, if available.
Following pairing, second-generation mysids produced in each compartment were counted, recorded and removed daily. Second-generation mysids were also observed for abnormal development and aberrant behavior. The test was terminated on Day 28, which was 9 days past the median time of the first brood release for the negative and solvent controls (Day 19). At test termination, the sex of each surviving first-generation mysid was confirmed microscopically and the total length of each mysid was measured using calipers. The mysids then were placed in a drying oven at approximately 60°C for approximately five days prior to measuring for dry weight data.

Statistical Analyses
Test endpoints analyzed statistically for first-generation mysids were survival, reproduction (the number of live young produced per reproductive day), and growth (total body length and dry weight). The results of the statistical analyses were used to aid in the determination of the NOEC, LOEC and MATC. However, scientific judgement was used to determine if statistical differences were biologically meaningful, and if the data followed a concentration-dependent response. The NOEC was defined as the highest test concentration that produced no significant treatment-related effects on survival, reproduction or growth. The LOEC was defined as the lowest test concentration that produced a significant treatment-related effect on survival, reproduction or growth. The MATC was calculated as the geometric mean of the NOEC and LOEC.
Data from the negative and solvent control groups for each parameter were compared using at test. Since no differences were detected between the two control groups (p > 0.05) for any of the parameters, the control data were pooled for comparison among the treatment groups.
Survival data was considered to be discrete-variable data, while reproduction and growth data were considered to be continuous-variable data. Discrete-variable data were analyzed using Chi-square and Fisher’s Exact tests to identify treatment groups that showed a statistically significant difference from the pooled control (p ≤ 0.05). All continuous-variable data were evaluated for normality using the Shapiro-Wilk’s test and for homogeneity of variance using Bartlett’s test (p = 0.01). The data for all
parameters passed the assumptions of normality and homogeneity of variance. Those treatments that were significantly different from the pooled control means were identified using either Bonferroni’s t-test or Dunnett’s test (p ≤ 0.05). All statistical tests were performed using a personal computer with TOXSTAT or SAS software.
Reference substance (positive control):
no
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
4.4 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: survival and growth
Key result
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
9.1 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: survival and growth
Key result
Duration:
28 d
Dose descriptor:
other: MATC
Effect conc.:
6.3 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: survival and growth
Details on results:
Measurement of Test Concentrations
Nominal concentrations selected for use in this study were 1.3, 2.5, 5.0, 10 and 20 μg /L. During the course of the test, the appearance of the test solution at these nominal concentrations was observed in both the diluter mixing chambers, where test substance stocks and dilution water were combined prior to delivery to the test chambers, and in the test chambers. Test solutions were observed for evidence of precipitate formation in the test system. The test solutions in the mixing chambers and test chambers appeared clear and colorless during the test, with no evidence of precipitation observed in any control or treatment solution.
Measured concentrations ranged from approximately 75 to 96% of nominal. With the exception of one sample from the 10 μg /L test concentration, measured concentrations ranged from approximately 82 to 103% of nominal. One sample from the 10 μg /L test concentration, collected on Day 18 of the test to guage the impact of a stock delivery error at this concentration, had a measured concentration that 173% of nominal. This measured concentration was representative of conditions during a brief period of the exposure, and was excluded from calculation of the mean measured concentration. When measured concentrations of the samples collected during the test were averaged, the mean measured test concentrations for this study were 1.2, 2.3, 4.4, 9.1 and 18 μg /L, representing 92, 92, 88, 91 and 90% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations.

Physical and Chemical Measurements of Water
Water temperatures were within the 25 ± 2°C range established for the test. Dissolved oxygen concentrations remained ≥74% of saturation (5.4 mg/L). Measurements of pH ranged from 7.9 to 8.1, and salinity ranged from 19 to 20‰ during the test. Light intensity at test initiation was 210 lux at the surface of the water of one representative test chamber.

Juvenile Survival to Pairing (Days 0 - 15)
After 15 days of exposure, survival of juvenile mysids in the negative and solvent control groups was 90 and 95%, respectively. Since there were no statistically significant differences in survival between the negative and solvent control groups (p > 0.05), the control data were pooled for comparisons to the treatment groups. Survival in the pooled control and in the 1.2, 2.3, 4.4, 9.1 and 18 μg /L treatment groups was 93, 95, 97, 97, 55 and 12%, respectively. Fisher’s Exact test indicated there was no statistically significant decreases in survival in treatment groups 1.2, 2.3 and 4.4 μg/L when compared to the pooled controls (p > 0.05). There were statistically significant decreases in survival in the 9.1 and 18 μg/L treatment groups when compared to the pooled controls (p ≤ 0.05). Consequently, the NOEC for juvenile survival (Day 0 –15) was 4.4 μg/L.
Surviving mysids in the control groups and in the 1.2, 2.3 and 4.4 μg/L treatment groups appeared normal during the period from test initiation to pairing on Day 15. There were observations of mysids that were small for the 1.2 μg/L treatment group in comparison to their cohorts. However, observations of small mysids in the 1.2 μg/L treatment group were infrequent, were not concentration-responsive and also occurred in the negative control group. Therefore, observations of small mysids in the 1.2 μg/L treatment group were not considered to be treatment related. Treatment-related signs of toxicity including lethargy and erratic swimming were observed for mysids in the 9.1 and 18 μg /L treatment groups. All surviving mysids in the 9.1 and 18 μg /L treatment groups were normal in appearance on Day 15 of the test.

Adult Survival After Pairing (Days 16 - 28)
Only seven mysids in the 18 μg/L treatment group survived until Day 15 of the test, the day of reproductive pair formation. Therefore, an insufficient number of mysids were available for pairing and the 18 μg/L treatment group was not continued into the reproduction phase of the study.
In general, mysids in the 1.2, 2.3, 4.4 and 9.1 μg /L treatment groups appeared normal after pairing, with no clinical signs of toxicity or mortality that was considered to be treatment related. At test termination, survival of adult mysids in the negative and solvent control groups was 94 and 98%, respectively. Since there were no statistically significant differences in survival between the negative and solvent control groups (p > 0.05), the control data were pooled for comparisons to the treatment groups. Survival in the pooled control and in the 1.2, 2.3, 4.4 and 9.1 μg /L treatment groups was 96, 96, 98, 96 and 97%, respectively. Fisher’s Exact test indicated there were no statistically significant decreases in survival in any treatment group when compared to the pooled controls (p > 0.05). Consequently, the NOEC for adult survival (Day 16 – 28) was 9.1 μg/L, the highest concentration tested during the adult phase of the study.

Reproduction
For each female, the number of reproductive days was defined as the number of days that the female was alive from the day of first brood release of any female in the test to the end of the test. The day of first brood release in this study was Day 16.
The mean number of young produced per reproductive day in the negative and solvent control groups were 0.98 and 0.92, respectively. Since there were no statistically significant differences in reproduction between the negative and solvent control groups (p > 0.05), the control data were pooled for comparisons to the treatment groups. The mean number of young produced per reproductive day in the pooled controls and in the 1.2, 2.3, 4.4 and 9.1 μg /L treatment groups was 0.95, 0.90, 0.81, 1.03 and 0.81, respectively. Bonferroni’s t-test indicated there were no statistically significant decreases in reproduction in any of the treatment groups in comparison to the pooled controls (p > 0.05). Consequently, the NOEC for reproduction was 9.1 μg/L, the highest concentration tested during the adult phase of the study.

Growth
Total length and dry weight mean measurements in the negative control group were 7.90 mm and 1.04 mg, respectively. In the solvent control group, total length and dry weight mean measurements were 7.89 mm and 1.09 mg, respectively. Since there were no
statistically significant differences (p > 0.05) between the negative and solvent control groups for either of the growth parameters, the control data were pooled for comparisons to the treatment groups.
The mean total lengths of mysids in the pooled controls and the 1.2, 2.3, 4.4 and 9.1 μg/L treatment groups were 7.90, 7.69, 7.59, 7.42 and 7.16 mm, respectively. The mean dry weights of mysids in the pooled controls and the 1.2, 2.3, 4.4 and 9.1 μg/L treatment groups were 1.06, 1.12, 1.15, 1.09 and 0.97 mg, respectively. When compared to the pooled controls, there was no statistically significant difference in mean length or dry weight for the 1.2 μg/L treatment group, or for mean dry weight among the 2.3 and 4.4
μg/L treatment groups. There were statistically significant decreases in mean total length in the 2.3, 4.4 and 9.1 μg/L treatment groups and in mean dry weight in the 9.1 μg/L treatment group, in comparison to the pooled controls (p ≤ 0.05). While there was a statistically significant decrease in the 2.3 and 4.4 μg/L treatment groups for mean total length, the difference was only 4 and 6%, respectively when compared to the pooled controls. Additionally, there was not a corresponding decrease in mean dry weight (a more sensitive measure of growth), or apparent impacts on reproduction or survival for the 2.3 and 4.4 μg/L treatment groups. Therefore, the slight reduction in mean length for the 2.3 and 4.4 μg/L treatment was not considered to be biologically significant. Consequently, the NOEC for growth (total length and dry weight) was 4.4 μg/L and the LOEC was 9.1 μg/L.
Reported statistics and error estimates:
see 'Details on results'
Validity criteria fulfilled:
yes
Conclusions:
Based on mean measured concentrations.
No-Observed-Effect Concentration:                           4.4 μg/L
Lowest-Observed-Effect Concentration:                     9.1 μg/L
Maximum Acceptable Toxicant Concentration:            6.3 μg/L
Executive summary:

The study was conducted under GLP and according to EPA OPPTS 850.1350 (Mysid Chronic Toxicity Test) and ASTM Standard E1191-03a.

Saltwater mysids (Americamysis bahia) were exposed to 14C-Ferbam at mean measured concentrations of 1.2 to 18 μg/L under flow-through conditions for 28 days, and were evaluated for survival, reproduction and growth. There were no significant, treatment-related reductions in survival or growth at concentrations <4.4 μg/L. There were also no significant reductions in reproduction at concentrations <9.1 μg/L. Growth, measured as total length and dry weight, and juvenile survival were the most sensitive biological endpoints measured. There was a statistically significant decrease in juvenile survival at the 9.1 and 18 μg/L test concentrations. Reproduction and growth were not evaluated for the 18 μg/L test concentration due to of low juvenile survival. There was a statistically significant, biologically meaningful decrease in length and dry weight among mysids at the 9.1 μg/L test concentration. Consequently, the NOEC, based on survival and growth, was 4.4 μg/L. The LOEC was 9.1 μg/L and the MATC was calculated to be 6.3 μg/L.

Description of key information

fresh water: according to GLP and OECD 202 / part II.

The 48 hours-EC50 values of the range-finding test was 0.097 mg/L (95% confidence limits = 0.08 -0.12 mg/L).

The 48 hours - EC50 values based on adult immobility with corresponding 95% confidence limits for the reference compound, potassium dichromate, was 0.56 mg/L (0.50 -0.64 mg/L), respectively.

At test termination the average cumulative daphnid young produced of the eight replicates and immobilization results showed were not significantly different (p=0.05) to 0.0028 mg/L (NOEC).

At test termination the average cumulative daphnid young produced of the eight replicates and immobilization results showed were significantly different (p=0.05) to 0.005 mg/L.

The MATC was 0.0037 mg/L.

marine water: under GLP and according to EPA OPPTS 850.1350 (Mysid Chronic Toxicity Test) and ASTM Standard E1191-03a

Based on mean measured concentrations.

No-Observed-Effect Concentration:                           4.4 µg/L

Lowest-Observed-Effect Concentration:                     9.1 µg/L

Maximum Acceptable Toxicant Concentration:            6.3 µg/L

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
0.003 mg/L
EC10, LC10 or NOEC for marine water invertebrates:
0.004 mg/L

Additional information

Key - freshwater: Perina, 1999

A 21-day chronic toxicity study of Ferbam to Daphnia magna was conducted according to GLP and OECD 202 / part II. The range-finding test was carried out in static system without renewal of test concentration for 48 hours. Twenty young daphnids (maximum 24 h-old) were divided into four groups of five organisms each, it were used at each test concentration and for controls. The nominal test concentration were: 0.032; 0.056; 0.1; 0.32 e 0.56 mg/L. The test flasks were incubated with light-dark cycle of 8 hours darkness and 16 hours light and the temperature was maintained 20 + 2°C during 48 hours. Based on EC50-48 hours of 0.097 mg/L the definitive chronic test was carried out in semi-static system with forty young daphnids (maximum 24 h-old) divided into eight groups of five organisms each, it were used at each test concentration and for the control. The nominal test concentration were controls; 0.0015; 0.0028; 0.005; 0.0009; 0.016; 0.03; 0.055 and 0.10 mg/L for evaluation survival and reproduction. Water quality parameters such as dissolved oxygen, conductivity, pH, hardness and alkalinity were measured throughout the test and were within acceptable limits.

From the data collected during this study, a 21-days EC50 was calculated to be 0.0180 mg/L (95% confidence limits = 0.02 - 0.02 mg/L) based on the tested concentration. At test termination the average cumulative daphnid young produced of the eight replicates and immobilization results showed were not significantly different (p=0.05) to 0.0028 mg/L (NOEC). At test termination the average cumulative daphnid young produced of the eight replicates and immobilization results showed were significantly different (p=0.05) to 0.005 mg/L. The MATC was 0.0037 mg/L. By the 8th day Daphnia at all exposure concentration had released their first brood in the water control.

As a quality check the daphnids were challenged with a reference compound, potassium dichromate, which 48-hours EC50 value was 0.56 mg/L.

Key - marinewater: Gallgahre, 2009

The study was conducted under GLP and according to EPA OPPTS 850.1350 (Mysid Chronic Toxicity Test) and ASTM Standard E1191-03a.

Saltwater mysids (Americamysis bahia) were exposed to 14C-Ferbam at mean measured concentrations of 1.2 to 18 μg/L under flow-through conditions for 28 days, and were evaluated for survival, reproduction and growth. There were no significant, treatment-related reductions in survival or growth at concentrations <4.4 μg/L. There were also no significant reductions in reproduction at concentrations <9.1 μg/L. Growth, measured as total length and dry weight, and juvenile survival were the most sensitive biological endpoints measured. There was a statistically significant decrease in juvenile survival at the 9.1 and 18 μg/L test concentrations. Reproduction and growth were not evaluated for the 18 μg/L test concentration due to of low juvenile survival. There was a statistically significant, biologically meaningful decrease in length and dry weight among mysids at the 9.1 μg/L test concentration. Consequently, the NOEC, based on survival and growth, was 4.4 μg/L. The LOEC was 9.1 μg/L and the MATC was calculated to be 6.3 μg/L.