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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:
31 Dec 2014 to 28 Jan 2015
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:
yes
Remarks:
Several deviations took place on the day before experimental termination, that are not considered to have a negative impact on the study.
Qualifier:
according to
Guideline:
other: EPA OPPTS 850.1000 Special Considerations for Conducting Aquatic Laboratory Studies
Version / remarks:
1996
Qualifier:
according to
Guideline:
other: ASTM Standard Guide for Conducting Life-Cycle Toxicity Tests with Saltwater Mysids
Version / remarks:
2008
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Prior to the start of the definitive exposure, samples were removed from one replicate of each treatment level and the control and analyzed for the test substance concentration. In addition, a sample of the stock solution was also analyzed during the pretest period. Results of the pretest analysis were used to judge whether sufficient quantities of test substance were being delivered and maintained in the exposure aquaria to initiate the definitive exposure. During the in-life phase of the definitive study, samples were removed from alternating replicate solutions of each treatment level and the control on days 0, 7, 14, 21 and 28 for analysis of test substance concentration. Samples of the stock solution were also analyzed at each of these intervals during the in-life phase of the definitive study. Three quality control (QC) samples were prepared at each sampling interval and remained with the set of exposure solution samples throughout the analytical process. Results of the analyses of the QC samples were used to judge the precision and quality control maintained during the analysis of exposure solution samples.
Vehicle:
no
Details on test solutions:
An 800 mg/L diluter stock solution was prepared prior to exposure initiation and as needed throughout the definitive exposure thereafter by adding, for example, 8.0058 g of test substance technical to approximately 1500 mL of deionized water. The resultant solution was sonicated for approximately 60 minutes before being diluted to a final volume of 10 L with deionized water and mixing using a magnetic stir bar and a stir plate. The resulting stock solutions were observed to be clear and tan in color. No undissolved test substance (e.g., precipitate) was observed in the diluter system (e.g., mixing chamber and chemical cells) or exposure solutions throughout the exposure period.
Test organisms (species):
Americamysis bahia (previous name: Mysidopsis bahia)
Details on test organisms:
TEST ORGANISM
- Common name: Saltwater mysid
- Source: The organisms were obtained from cultures maintained at Smithers Viscient. The brood stock originated from MBL Aquaculture located in Sarasota, Florida, and had been maintained in house in continuous culture for approximately 36 months prior to use.
- Age: <24 hours old
- Feeding during test: Yes, see 'Details on test conditions'.
- Health before exposure: The culture organisms did not show any sign of sickness, disease, injuries or abnormalities from the day of receipt to the day of exposure initiation. Brood stock and test organisms were determined to be in good health at the start of the exposure phase.

ISOLATION
Adult, gravid female mysids were isolated from the brood stock the day prior to initiation and held in isolation. The exposure was then initiated ≤ 24 hours from this isolation with young collected from the isolated females.

CULTURING
Mysids were cultured in six recirculating 80-L glass aquaria containing dilute, natural seawater. Standard aquarium under gravel filters were used to provide aeration and a current conducive to feeding. The seawater in the aquaria was characterized as having a salinity of 22‰, a dissolved oxygen range of 90.3 to 96.9% of saturation, and a pH range of 7.8 to 7.9 during the 14-day period prior to exposure initiation. Brood stock and test organisms were cultured and tested in seawater from the same source. The area in which the mysids were cultured received a regulated photoperiod of 16 hours of light at an intensity range of 24 to 52 footcandles and 8 hours of darkness. A commercial aquarium heater was used to maintain culture solution temperature at 26 °C.
Test type:
flow-through
Water media type:
saltwater
Limit test:
no
Total exposure duration:
28 d
Test temperature:
25 - 27 °C
pH:
7.3 - 8.1
Dissolved oxygen:
5.03 - 7.01 mg O2/L (70.5 - 96.6% oxygen saturation)
Salinity:
2.0 - 2.1%
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 0.13, 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L
- Mean measured concentrations:
Details on test conditions:
EXPOSURE SYSTEM
Prior to exposure initiation, an FMI Pump was calibrated to deliver 0.0099 L/cycle of the 800 mg/L diluter stock solution into the diluter system's chemical mixing chamber, which also received 1.97 L of dilution water per cycle. The mixing chamber was positioned over a water-driven magnetic stir plate. The continuous stirring aided the solubilization of the test substance with the dilution water. The solution in the mixing chamber was equivalent to that of the highest nominal test concentration and was proportionally diluted (50%) to produce the remaining nominal test concentrations.
A set of negative control vessels was also established which contained the same dilution water and was maintained under the same conditions as the treatment level vessels, but contained no test substance.
The life-cycle toxicity test was conducted using an exposure system consisting of an intermittent-flow proportional diluter (Mount and Brungs, 1967), a temperature-controlled water bath, and a set of 28 exposure aquaria. Each glass test aquarium measured 30 x 15 x 20 cm with a 10-cm high side drain that maintained a constant exposure solution volume of approximately 4.5 L. Four replicates were maintained for all treatments and the control. The exposure system was designed to provide six concentrations of the test substance and a dilution water control. The exposure system was in proper operation for seven days prior to exposure initiation to allow equilibration of the test substance in the diluter apparatus and exposure vessels. The exposure system was labeled with the appropriate study number and exposure vessels were labelled to identify the nominal test substance concentration and designated replicate.
During each cycle of the diluter system, approximately 250 mL of exposure solution was delivered to each replicate test vessel with a flow-splitting accuracy of 5%. During the study, the diluter provided the exposure solutions to each test vessel at a rate of approximately 7.7 aquarium volume additions per day to provide a 90% test solution replacement rate of approximately 7 hours (Sprague, 1969). The function of the diluter system (e.g., dilution water flow rates) was monitored daily and a complete check was performed twice each day. In addition, weekly analysis of the exposure solutions was used to verify proper operation of the diluter system.
For the first 12 days of exposure, each exposure aquarium contained one retention chamber. The retention chambers, used to retain sexually immature mysids, were constructed of glass petri dishes, 10 cm in diameter, 2 cm deep, to which a 14 cm high Nitex® screen collar (350-μm mesh size opening) was attached with silicone sealant. The solution volume within each retention chamber was approximately 785 mL.
Once all mysids appeared to be sexually mature (test day 13), male and female pairs were transferred to separate pairing chambers. Following this distribution, each exposure aquarium contained one retention chamber and a maximum of five pairing chambers. Pairing chambers, used to retain sexually mature male and female organisms, were constructed of 6-cm diameter petri dishes, 1.5 cm deep, to which a 14 cm high Nitex® screen collar (350-μm mesh size opening) was attached with silicone sealant. Solution volume within each pairing chamber was approximately 250 mL.

TEST CONDITIONS
The test area was illuminated with Sylvania Octron fluorescent bulbs at an intensity range of 29 to 56 footcandles (320 to 600 lux), measured during the in-life exposure, and a photoperiod of 16 hours light and 8 hours darkness with a 30-minute transition period. This photoperiod is not the recommended photoperiod in the OCSPP guidance (U.S. EPA, 1996a); however, this photoperiod is supported by ASTM Standard Guide 1191-03a (ASTM, 2008). The transition period allowed gradual acclimation to changing light intensity, thereby reducing stress from sudden changes in light intensity on the test organisms. Light intensity was measured using a VWR light meter. The study was conducted in a water bath designed to maintain the test solution temperatures at 25 ± 2 °C. Two Process Technology USA Immersion Heaters regulated by an Omega CN 76000 temperature controller were used to maintain test solution temperature.

F0 LIFE-CYCLE EXPOSURE INITIATION
Mysids, ≤ 24 hours old, were divided among 28 beakers. The beakers contained dilution water and were held in a water bath maintained at approximately 25 ºC. The organisms were impartially selected and distributed to the beakers by adding five organisms at a time to each beaker until all beakers contained five mysids. This process was repeated until each beaker contained 20 mysids. A random number generator was used to randomly assign each beaker to 28 labelled retention chambers. Each group of 20 mysids was then transferred to the beaker’s randomly assigned retention chamber. The exposure was initiated when the retention chambers were placed in their respective exposure aquaria. Each exposure aquarium contained one retention chamber, yielding 20 mysids per replicate vessel and 80 organisms for each treatment level and the control.

F0 LIFE-CYCLE MYSID PAIRING
The length of time for brood appearance (i.e., gravid females) was noted for all first generation mysids prior to pairing (approximately day 12). When sexual maturity was reached (day 13) one mature male and one mature female were randomly assigned to each of the pairing chambers (with a maximum of five male/female pairs per replicate). Unpaired mysids were pooled and maintained in one of the initial retention chambers until they were paired or until test termination. When three or fewer unpaired mysids remained in a retention chamber, they were transferred to a separate pairing chamber, to conserve space and reduce the overall surface area within the exposure vessel. Male mysids from this pool were used to replace dead males from the paired (male/female) groups. Females that died in the pairing chambers were replaced.

MYSID COUNTING AND BIOLOGICAL OBSERVATIONS
In order to observe the mysids during the exposure period, each retention chamber was gently lifted from the aquaria daily. The chamber was then placed into a Pyrex® dish containing dilution water and the dish was placed on a light table. During this procedure, the water level in each chamber was reduced by allowing water to drain through the screen. The numbers of dead and living organisms were counted and any abnormal appearance or behavior was recorded. Due to the rapid movement of mysids in a single chamber containing up to 20 mysids, survival of the test organisms was estimated for the first 12 days of the test, i.e., prior to pairing of the mysids. After males and females had been paired (day 13) actual counts of survival were made and the number of dead males and females, the number of offspring produced by each individual female and any abnormal appearance or behavior was recorded. Observations were made daily throughout the study with the exception of day 27. Dead parental mysids and offspring were recorded, removed, and discarded when observed during the test. Mortality was defined as lack of movement after gentle prodding with a glass pipette. Following the observation period, retention chambers were brushed clean, debris was removed, and they were returned to the respective aquaria.

F1 INITIATION
During the reproductive phase, groups of 10 offspring per replicate, 40 per treatment (if 10 offspring could not be collected, a reduced number of young was collected and evaluated; n > 5) were removed from mysid chambers in each replicate vessel, pooled and placed in a separate pairing chamber in that replicate. Chambers were established based on the number of available juvenile F1 mysids; therefore, each chamber was not necessarily initiated on the same day. One F1 group was established and monitored for each replicate vessel where reproduction was sufficient. The chambers with F1 mysids were established to monitor survival 96 hours post-release. This observation period ensured an equal observation period across all treatment levels and the control prior to exposure termination.

F1 BIOLOGICAL OBSERVATIONS AND SURVIVAL
At the time an F1 generation pairing chamber was established and daily thereafter for 96 hours, observations of stress, abnormal behavior (including discoloration, immobilization and inability to maintain position in the water column), and survival were made. Dead mysids (defined as the absence of mobility and failure to respond to gentle prodding) were recorded and removed from each replicate test vessel daily. Missing mysids were considered dead.

FEEDING
Throughout the test, mysids were fed live brine shrimp (Artemia salina) nauplii, ≤ 48 hours old (post-hydration), twice daily. At least one of these feedings was with brine shrimp nauplii enriched with Selco®, a substance high in saturated fatty acids. Mysid survival, growth and reproduction are improved with high levels of Ω-3 highly unsaturated fatty acids. Selco® is added to newly hatched brine shrimp and they become enriched by feeding on the lipid-coated microparticles. Densities per mL of concentrated collections of nauplii from Artemia cultures created from daily hydrated cysts were determined by triplicate counts of aliquots pipetted onto filter paper. Based on these densities, various volumes of nauplii were fed to mysids per feeding in accordance with the following regime: rations for F0 retention chambers (containing 20 mysids each) were approximately 90 nauplii/mysid on test days 0 to 3, 135 nauplii/mysid on test days 4 to 6, 180 nauplii/mysid on test days 7 to 9 and 225 nauplii/mysid on test day 10 to pairing. From day of pairing until test termination, the F0 pairing chambers were fed approximately 450 nauplii/mysid and the retention chambers were fed approximately 3600 nauplii/chamber. After F1 chambers were initiated, chambers were fed a ration of approximately 90 nauplii/mysid. All treatments and the control received, as near as reasonably possible, the same ration of food. Excess brine shrimp and organic debris in the test chambers was removed prior to the addition of new nauplii. Prior to use of a new batch of feed, a representative sample of the food source was analyzed for the presence of pesticides, PCBs, and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds were detected at concentrations that are considered toxic in any of the food samples analyzed in agreement with ASTM (2002) standard practice. Based on the analysis for pesticides and the performance of the control organisms, the food source was considered to be of acceptable quality since all analyte concentrations were below levels of concern and control organisms exceeded guideline criteria.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The seawater was pumped under constant pressure through PVC pipes to the intermittent-flow proportional diluter system. The seawater used for this study had a salinity range of 19 to 21‰ and a pH range of 7.7 to 7.9. Routine analyses were conducted periodically on representative samples of the water source for the presence of pesticides, PCBs, and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds were detected at concentrations that are considered toxic in any of the water samples analyzed in agreement with ASTM (2002) standard practice. Based on these analyses and the performance of the control organisms, the water source was considered to be of acceptable quality since all analyte concentrations were below levels of concern and control organisms exceeded guideline performance criteria. Representative samples of the dilution water were analyzed monthly for total organic carbon (TOC) concentration. The TOC concentration of the dilution water was 1.1 and 1.2 mg/L for December 2014 and January 2015, respectively.
- Culture medium different from test medium: Same

WATER QUALITY MEASUREMENTS
Temperature, dissolved oxygen concentration, pH and salinity were measured in each replicate on day 0 and alternated between replicates daily thereafter throughout the exposure period, for each treatment level and the control. In addition, exposure solution temperature was continuously monitored in one control vessel (replicate A) using a VWR minimum/maximum thermometer. Salinity concentration was measured with a Yellow Springs Instrument (YSI) Model Pro30 conductivity/salinity meter. The dissolved oxygen concentration and daily temperature were measured using a YSI Model 550A dissolved oxygen meter/temperature probe. A YSI Model pH100A pH meter was used for pH measurements.

BIOMASS LOADING RATE
During the 28-day exposure period, maximum biomass loading did not exceed 0.0026 g/L flowing solution per day or 0.020 g/L of solution at any time, in any replicate exposure aquarium. Calculations of biomass loading are based on conservative culture estimate of the typical average wet weight of an adult mysid (0.0045 g).

EFFECT PARAMETERS MEASURED: survival, sub-lethal effects, body length, dry weight and reproductive success
- Survival and sublethal effects: At the time an F1 generation pairing chamber was established and daily thereafter for 96 hours, observations of stress, abnormal behavior (including discoloration, immobilization and inability to maintain position in the water column), and survival were made. Dead mysids (defined as the absence of mobility and failure to respond to gentle prodding) were recorded and removed from each replicate test vessel daily. Missing mysids were considered dead.
- Mean total body length and dry weight: At test termination, all mysids were euthanized by immersion in ice-cold, deionized water. The mysids were carefully removed, blotted dry on absorbent paper, and separated into male and female groups for each replicate exposure level. A digital photograph was then taken of each mysid using a binocular dissection microscope for individual body length measurements. Individual body length was measured to the nearest 0.01 mm. Images were obtained using the following hardware and software: Zeiss Stemi-2000 CS, a Zeiss AxioCam ICc 5 and Zeiss Zen image analyzing software. Following these procedures, male and female mysids were transferred to aluminum pans, dried in an oven at ~100 °C for 20.5 hours and then placed in a desiccator. Individual dry body weight to the nearest 0.01 mg was determined using an analytical balance. Individual lengths and weights of all surviving males and females were recorded separately for each replicate of each concentration and the control.
- Reproductive success: Reproductive success was calculated for each replicate aquarium (treatments and the control) as the total number of offspring produced per female. In addition, the percentage of actively reproducing females in each replicate of each treatment and the control was determined.

PRELIMINARY TEST
A preliminary 28-day flow-through exposure was conducted at Smithers Viscient exposing juvenile mysids (40 mysids per treatment level; 20 per replicate) to nominal the test substance concentrations of 0.031, 0.063, 0.13, 0.25 and 0.50 mg/L, and a control. The procedures used for this exposure were similar to the procedures used for the definitive exposure.
- Results used to determine the conditions for the definitive study: F1 generation 96-hour post-release survival among control mysids averaged 95%. F1 generation 96-hour post-release survival in the treatment levels tested (0.031, 0.063, 0.13, 0.25 and 0.50 mg/L) averaged 85, 95, 95, 100 and 80%, respectively. Based on these results, the nominal concentrations selected for the definitive exposure were 0.13, 0.25, 0.50, 1.0 2.0 and 4.0 mg/L.
Reference substance (positive control):
no
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
0.56 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: most sensitive endpoint
Remarks:
additional effect values are presented in 'Any other information on results incl. tables'
Duration:
28 d
Dose descriptor:
LOEC
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: most sensitive endpoint
Details on results:
Results on survival, reproductive succes, growth and F1 survival are tabulated in 'Any other information on materials and methods incl. tables'.

SURVIVAL
- Mean survival males: At test termination, post-pairing, mean survival of 91% was observed among male mysids in the control. Mean survival of 87, 93, 83, 86, 62 and 79% was observed among male mysids exposed to the 0.14, 0.24, 0.56, 1.1, 2.0 and 3.9 mg/L treatment levels, respectively. Williams Multiple Comparison test determined a significant difference in male survival among organisms exposed to the 2.0 and 3.9 mg/L treatment levels compared to the control data.
- Mean survival females: At test termination, post-pairing, mean survival of 89% was observed among female mysids in the control. Mean survival of 96, 97, 96, 85, 93 and 100% was observed among female mysids exposed to the 0.14, 0.24, 0.56, 1.1, 2.0 and 3.9 mg/L treatment levels, respectively. Williams Multiple Comparison Test determined no significant difference in female survival among organisms exposed to any of the treatment levels tested compared to the control data.
- Following 28 days of exposure, overall, mean survival of 79% was observed among control mysids. Mean survival of 79, 87, 74, 68, 56 and 34% was observed among mysids exposed to the 0.14, 0.24, 0.56, 1.1, 2.0 and 3.9 mg/L treatment levels, respectively. Williams Multiple Comparison Test determined a significant difference in survival among organisms exposed to the 1.1, 2.0 and 3.9 mg/L treatment levels compared to the control data.

OFFSPRING
At test termination, the mean number of offspring per female for organisms in the control was 13.0. The mean number of offspring per female was 13.8, 15.7, 12.0, 15.7, 15.8 and 6.4 among mysids exposed to the 0.14, 0.24, 0.56, 1.1, 2.0 and 3.9 mg/L treatment levels, respectively. Williams Multiple Comparison Test determined a significant difference in the mean number of offspring per female among organisms exposed to the 3.9 mg/L treatment level compared to the control data.

GROWTH
- Mean total body length males: The mean total body length of male mysids in the control was 6.84 mm. The mean total body length of male mysids exposed to the 0.14, 0.24, 0.56, 1.1, 2.0 and 3.9 mg/L treatment levels was 6.89, 6.83, 6.88, 7.02, 6.91 and 7.15 mm, respectively. Williams Multiple Comparison Test determined no significant difference in total body length of male mysids among organisms exposed to any of the treatment levels tested compared to the control data.
- Mean total body length females: The mean total body length of female mysids in the control was 6.89 mm. The mean total body length of female mysids exposed to the 0.14, 0.24, 0.56, 1.1, 2.0 and 3.9 mg/L treatment levels was 6.97, 7.27, 7.23, 7.39, 7.44 and 7.64 mm, respectively. Williams Multiple Comparison Test determined no significant difference in total body length of female mysids among organisms exposed to any of the treatment levels tested compared to the control data.
- Mean dry body weight males: The mean dry body weight of male mysids in the control was 0.62 mg. The mean dry body weight of male mysids exposed to the 0.14, 0.24, 0.56, 1.1, 2.0 and 3.9 mg/L treatment levels was 0.67, 0.63, 0.65, 0.66, 0.64 and 0.79 mg, respectively. Jonckheere-Terpstra Step-Down Test determined no significant difference in the dry body weight of male mysids exposed to any of the treatment levels tested compared to the control data.
- Mean dry body weight males: The mean dry body weight of female mysids in the control was 0.83 mg. The mean dry body weight of female mysids exposed to the 0.14, 0.24, 0.56, 1.1, 2.0 and 3.9 mg/L treatment levels was 0.92, 0.98, 0.99, 0.99, 0.92 and 0.89 mg, respectively. Williams Multiple Comparison Test determined no significant difference in the dry body weight of female mysids exposed to any of the treatment levels tested compared to the control data.

F1 96-H SURVIVAL
Following the 96-hour observation period, mean percent survival of 98% was observed among F1 mysids in the control. Mean percent survival of 93, 100, 95, 95, 98 and 100% was observed among F1 mysids exposed to the 0.14, 0.24, 0.56, 1.1, 2.0 and 3.9 mg/L treatment levels, respectively. The F1 mysids in replicates C and D of the 3.9 mg/L treatment level were inadvertently discontinued at 72 hours; therefore, this treatment level was excluded from statistical analysis of F1 survival. Williams Multiple Comparison Test determined no significant difference in F1 mysid survival among organisms exposed to any of the treatment levels statistically analyzed compared to the control data.
Reported statistics and error estimates:
See 'Any other information on materials and method incl. tables for a detailed description of the statistical analysis'

 Table: Mysid (Americamysis bahia) life-cycle exposure to the test substance – First generation (F0) survival

Mean Measured Concentration (mg/L)

Male Survival(a) (%)

Female Survival(a) (%)

Post-Pairing Survival

(%)

28-Day

Survival(b)

(%)

Control

A

88

83

86

75

B

89

83

87

76

C

86

90

88

79

D

100

100

92

86

Mean (SD)(c)

91 (6)

89 (8)

88 (3)

79 (5)

0.14

A

75

100

88

78

B

88

100

83

79

C

86

100

93

72

D

100

86

94

88

Mean (SD)

87 (10)

96 (7)

89 (5)

79 (7)

0.24

A

90

88

89

84

B

100

100

100

94

C

100

100

100

94

D

83

100

88

74

Mean (SD)

93 (8)

97 (6)

94 (7)

87 (10)

0.56

A

100

100

100

82

B

80

100

92

73

C

63

100

81

72

D

88

83

86

67

Mean (SD)

83 (16)

96 (8)

90 (8)

74 (6)

1.1

A

100

100

100

71

B

83

67

69

61

C

83

100

80

71

D

78

75

68

68

Mean (SD)

86 (10)

85 (17)

79 (15)

68(d) (4)

2.0

A

86

100

92

71

B

80

80

80

44

C

43

90

71

63

D

40

100

73

47

Mean (SD)

62(d) (24)

93 (10)

79 (10)

56(d) (13)

3.9

A

100

100

100

26

B

67

100

89

47

C

50

100

86

32

D

100

100

100

30

Mean (SD)

79(d) (25)

100 (0)

94 (7)

34(d) (9)

 

a)Calculations of male and female survival began after pairing.

b) 28 day survival encompasses all mortalities from day 0 through termination, regardless of pairing status.

c) Mean values are presented with standard deviations (SD) in parentheses.

d) Significantly reduced compared to the control, based on Williams Multiple Comparison Test

NOTE: Values presented have been rounded; however, statistical analysis was performed using unrounded values.

 

Table: Mysid (Americamysis bahia) life-cycle exposure to the test substance – First generation (F0) reproductive success (offspring per female)

Mean Measured

Concentration

(mg/L)

Percent of

Females

Producing Young

Number of Offspring

Per Female

Control

A

100

17.4

B

100

11.0

C

100

14.6

D

100

8.8

Mean (SD)(a)

100 (0)

13.0 (3.8)

0.14

A

100

13.2

B

100

13.2

C

100

15.0

D

100

13.8

Mean (SD)

100 (0)

13.8 (0.8)

0.24

A

100

19.8

B

100

14.8

C

100

15.4

D

80

12.8

Mean (SD)

95 (10)

15.7 (3.0)

0.56

A

80

10.6

B

80

9.6

C

100

12.4

D

100

15.4

Mean (SD)

90 (11.5)

12.0 (2.5)

1.1

A

80

11.4

B

100

13.0

C

100

20.0

D

100

18.2

Mean (SD)

95 (10)

15.7 (4.1)

2.0

A

100

17.6

B

80

13.0

C

80

14.8

D

100

17.6

Mean (SD)

90 (11.5)

15.8 (2.3)

3.9

A

100

11.0

B

100

13.4

C

0

0.0

D

50

1.0

Mean (SD)

63 (47.9)

6.4(b) (6.8)

a) Mean values are presented with standard deviations (SD) in parentheses.

b) Significantly reduced compared to the control, based on Williams Multiple Comparison Test

NOTE: Values presented have been rounded; however, statistical analysis was performed using unrounded values.

 

Table: Mysid (Americamysis bahia) life-cycle exposure to the test substance – First generation (F0) male and female total body length

Mean Measured Concentration (mg/L)

Mean Total Body Weight

Males

Females

Control

A

6.92

6.97

B

6.81

6.91

C

7.03

6.89

D

6.57

6.80

Mean (SD)(a)

6.84 (0.20)

6.89 (0.07)

0.14

A

6.92

7.01

B

6.82

6.99

C

7.05

6.98

D

6.78

6.90

Mean (SD)

6.89 (0.12)

6.97 (0.05)

0.24

A

6.94

7.42

B

6.76

7.04

C

6.76

7.14

D

6.88

7.47

Mean (SD)

6.83 (0.09)

7.27 (0.21)

0.56

A

6.75

7.11

B

7.05

7.23

C

6.82

7.46

D

6.90

7.12

Mean (SD)

6.88 (0.13)

7.23 (0.16)

1.1

A

6.82

7.25

B

6.91

7.43

C

7.12

7.43

D

7.21

7.43

Mean (SD)

7.02 (0.18)

7.39 (0.09)

2.0

A

7.10

7.44

B

6.64

7.41

C

7.09

7.42

D

6.79

7.48

Mean (SD)

6.91 (0.23)

7.44 (0.03)

3.9

A

6.94

7.69

B

7.12

7.37

C

7.20

7.66

D

7.34

7.86

Mean (SD)

7.15 (0.17)

7.64 (0.20)

a) Mean values are presented with standard deviations (SD) in parentheses.

 

Table: Mysid (Americamysis bahia) life-cycle exposure to the test substance – First generation (F0) male and female dry body weight

Mean Measured

Concentration

(mg/L)

Dry Body Weight (mg)

 

Males

Females

Control

A

0.65

0.89

B

0.65

0.82

C

0.63

0.86

D

0.55

0.76

Mean (SD)(a)

0.62 (0.05)

0.83 (0.06)

0.14

A

0.68

0.89

B

0.58

0.85

C

0.70

1.02

D

0.69

0.93

Mean (SD)

0.67 (0.06)

0.92 (0.07)

0.24

A

0.62

0.96

B

0.59

0.84

C

0.67

0.90

D

0.65

1.24

Mean (SD)

0.63 (0.04)

0.98 (0.18)

0.56

A

0.67

0.97

B

0.66

0.93

C

0.60

1.03

D

0.67

1.04

Mean (SD)

0.65 (0.04)

0.99 (0.05)

1.1

A

0.67

0.93

B

0.62

0.92

C

0.66

1.00

D

0.69

1.11

Mean (SD)

0.66 (0.03)

0.99 (0.09)

2.0

A

0.70

0.88

B

0.58

0.95

C

0.73

0.93

D

0.57

0.94

Mean (SD)

0.64 (0.08)

0.92 (0.03)

3.9

A

0.65

0.99

B

0.63

0.87

C

0.74

0.79

D

1.13

0.91

Mean (SD)

0.79 (0.23)

0.89 (0.08)

a) Mean values are presented with standard deviations (SD) in parentheses.

NOTE: Values presented have been rounded; however, statistical analysis was performed using unrounded values.

 

Table: Mysid (Americamysis bahia) life-cycle exposure to the test substance – Second generation (F1) survival at 96-hours post-release

Mean Measured

Concentration

(mg/L)

Survival (%)

 

Control

A

0.9

B

1

C

1

D

1

Mean (SD)(a)

98 (5)

0.14

A

1

B

0.9

C

1

D

0.8

Mean (SD)

93 (10)

0.24

A

1

B

1

C

1

D

1

Mean (SD)

100 (0)

0.56

A

1

B

0.9

C

0.9

D

1

Mean (SD)

95 (6)

1.1

A

1

B

0.8

C

1

D

1

Mean (SD)

95 (10)

2.0

A

0.9

B

1

C

1

D

1

Mean (SD)

98 (5)

3.9

A

1

B

1

C

NA(b)

D

NA

Mean (SD)

100 (0)

a) Mean values are presented with standard deviations (SD) in parentheses.

b) NA = Not Applicable. F1 chamber was inadvertently discontinued at 72 hours; therefore, this replicate was excluded from statistical analysis of F1 survival (see protocol deviation).

NOTE: Values presented have been rounded; however, statistical analysis was performed using unrounded values.

Table: The NOEC and LOEC of all endpoints

Measured response

Based on Mean Measured Concentrations (mg/L)

NOEC

LOEC

28-d Survival

0.56

1.1

F1 Survival

3.9

>3.9

Female Dry Weight

3.9

>3.9

Female Survival

3.9

>3.9

Female Total Length

3.9

>3.9

Male Dry Weight

3.9

>3.9

Male Survival

1.1

2.0

Male Total Length

3.9

>3.9

Offspring Per Female

2.0

3.9

Validity criteria fulfilled:
yes
Remarks:
See 'Any other information on materials and methods incl. tables'
Conclusions:
The 28-d NOEC value for saltwater invertebrates (Americamysis bahia) is 0.56 mg/L based on the most sensitive endpoint survival.
Executive summary:

A life-cycle toxicity test with the test substance was conducted in accordance with OCSPP Draft Guideline 850.1350 (1996) and in compliance with GLP. This study was performed to determine the chronic (full life-cycle) toxicity of the test substance to the mysid, Americamysis bahia, under flow-through conditions. 28-Day survival, male and female survival (post-pairing), reproduction (based on average young produced per female) and male and female growth (mean total body length and dry weight) were measured as test endpoints. The life-cycle toxicity test was conducted using an exposure system consisting of an intermittent-flow proportional diluter (Mount and Brungs, 1967), a temperature-controlled water bath, and a set of 28 exposure aquaria. Four replicates were maintained for all treatments (i.e., 0.13, 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L nominal, 0.14, 0.24, 0.56, 1.1, 2.0 and 3.9 mg/L mean measured) and the control. The test area was illuminated at an intensity range of 29 to 56 footcandles (320 to 600 lux), measured during the in-life exposure, and a photoperiod of 16 hours light and 8 hours darkness with a 30-minute transition period. The study was conducted in a water bath designed to maintain the test solution temperatures at 25 ± 2°C. During the in-life phase of the definitive study, samples were removed from alternating replicate solutions of each treatment level and the control on days 0, 7, 14, 21 and 28 for analysis of the test substance concentration. Samples of the stock solution were also analyzed at each of these intervals during the in-life phase of the definitive study. Based on mean measured concentrations of the test substance and the most sensitive indicator (i.e. 28 day survival), the No-Observed-Effect Concentration (NOEC) was determined to be 0.56 mg/L. The Lowest-Observed-Effect Concentration (LOEC) based on 28-d survival for mysids was determined to be 1.1 mg/L. Exposure to the test substance did not cause a reduction in length or weight of male and female mysid at the concentrations tested. Statistically significant reductions in the number of offspring was only observed in the highest treatment level (i.e. 3.9 mg/L).

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 Jun to 14 Jul 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guidance Document “Freshwater Lentic Field Tests”
Version / remarks:
2006
Deviations:
no
Qualifier:
according to
Guideline:
other: SETAC-EUROPE, Guidance Document on Testing Procedures for Pesticides in Freshwater Static Mesocosms
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
other: HARAP (Campbell et al., 1999) Guidance Document on Higher Tier Aquatic Risk Assessment for Pesticides
Version / remarks:
1999
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- The concentrations of the active substance were regularly monitored to calculate the doses needed to maintain the water concentrations. The water was also analysed for the insecticidally active plant and soil metabolite of the test item due to its toxicity to aquatic invertebrates.
- Water samples were taken just before each application (except for the first one), three hours after, and two, three and / or four days after each application until one day before the next application in order to measure the concentration of the a.s. in the water column and to calculate the doses needed to maintain the concentrations.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Application method: Directly into the water column by means of a specially adapted separating funnel

Test organisms (species):
other: Cloeon dipterum
Test type:
other: outdoor mesocosms representative of a typical pond community
Water media type:
freshwater
Limit test:
no
Total exposure duration:
35 d
Hardness:
no measurement reported
Test temperature:
- Environment: 10 - 35 °C
- Water temperature: 14 - 21 °C
pH:
7.7 - 9.9
Dissolved oxygen:
no measurement reported
Salinity:
no measurement reported
Conductivity:
no measurement reported
Nominal and measured concentrations:
- Nominal concentrations: 0.0 (control), 0.1, 0.3, 1.0, 3.0, 10 μg a.s./L (corresponding to 0.0 (control), 0.4, 1.2, 4.0, 12, 40 μg test material/L).
- Measured concentrations: 80 - 100 % of the nominal concentrations.
The absolute minimum and maximum measured test item concentration in 420 analyzed samples were 45 and 135 % of the nominal concentration. However, deviations from the 80 to 120 % range were rare. The relative mean measured concentration over all samples was 100 % of nominal. Time weighted average concentrations were 101 % of nominal, ranging from 93 to 108 % over the 15 enclosures treated and from 97 to 105 % for the mean TWA per treatment level. No test item was detected in the samples of the controls (all values < LOQ of 0.05 μg/L).
Details on test conditions:
TEST SYSTEM
- Mesocosm description: Stainless steel enclosures in a large lined basin with a diameter of approximately 9.39 m and a volume of about 76,000 L; filled with a clay layer of approximately 10 cm; Sediment was collected from a depth of approximately 0.5 m below the water surface from a small lake on site and spread onto the layer of clay to build a sediment layer of about 10 cm
- Mesocosm water: Water was originally taken in July 2014 from a lake on site with no known history of chemical contamination. The water was mixed with rain to give the ultimate water body.
- Enclosure: Stainless steel enclosures were pressed into the sediment. Each enclosure had a diameter of approximately 143 cm (surface area approximately 1.60 m2) and a depth of approximately 150 cm. With a depth of the water body of about 100 cm ± 20 %, the total volume of each enclosure was approximately 1600 L.
- Location of enclosure: Twenty stainless steel enclosures installed in a large artificial pond located at the test site were used for the test. The test systems contained indigenous flora and fauna (i.e., algae, macrophytes, zooplankton and macroinvertebrates) which originated from the sediments, water and aerial colonisation by flying insects.
- Number of replicates per concentration: the nominal water concentrations were tested in three replicate enclosures while five enclosures served as untreated controls
- Application in the water column: The test solutions were directly applied into the water column by means of a specially adapted separating funnel


MONITORING
- Effects on larvae and emergence of mayflies (Cloeon sp.) were monitored twice before and over the course of 35 days after the first application. Mayfly abundance data (larvae and emerged adults) were analysed by means of the multiple t-test of Williams for significant difference to the controls, for each sampling date.
- Phytoplankton (chlorophyll a), zooplankton and macroinvertebrates were sampled twice before the exposure period to characterize the community. Water parameters, macrophyte area coverage and periphyton abundance (taxonomic identification and cell counts) were monitored before and also during the exposure.
- Water samples were taken just before each application (except for the first one), three hours after, and two, three and / or four days after each application until one day before the next application in order to measure the concentration of the a.s. in the water column and to calculate the doses needed to maintain the concentrations
- Mayfly larvae were sampled and counted once per week. Two baskets with stones and the shoots of the macrophyte Ceratophyllum demersum as substrate were installed about 20 and 60 cm beneath the water surface and remained in-situ the week between sampling to allow recolonization.
- Emergence of mayflies was measured by means of two emergence traps per enclosure, situated over the same structures (one over open water and one over macrophytes) in each enclosure. The emergence traps were stainless steel constructions with a trapping device containing a sample fixative at the apex of the structure. The diameter of the traps is approx. 40 cm (area 0.126 m²).
- Macroinvertebrates other than mayflies, zooplankton, phytoplankton, periphyton and macrophytes were assessed twice before the exposure period in order to characterize the enclosures. Periphyton and area coverage by macrophytes were also assessed once during the exposure period due to their relevance as food source or habitat for the mayflies.

ENVIRONMENTAL TEST CONDITIONS RANGES
- Water level: 97.6 to 99.5 cm
- Cloudiness: Between 0 to 100%
Reference substance (positive control):
no
Key result
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
0.3 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: larval abundance and emergence of Cloeon dipterum
Details on results:
RESULTS PER SPECIES
- Zooplankton: In total, 32 taxa/stages belonging to three different classes (crustacea, insecta, rotatoria) were identified in the 40 zooplankton samples taken before the exposure period. Cyclopoida adults and copepodids were pooled for calculation of the diversity indices. On average 13 taxa and 745 individuals/L were found in the zooplankton samples.
- Macroinvertebrates: Thirty-nine macroinvertebrate taxa or stages were differentiated in artificial substrate samplers before start of exposure. For calculation of diversity indices, different stages of the same taxon or taxa which potentially overlap were pooled. On average, 14 Taxa and 298 macroinvertebrate individuals were found per sample. The macroinvertebrates were clearly dominated by insects.
- Phytoplankton: Before the exposure period, phytoplankton abundance was analysed by delayed fluorescence measurements of chlorophyll a concentrations. On average, the total chlorophyll a concentration was 7 μg/L. Almost 50 % of this was attributed to Cryptophytes and 2 μg/L to Chromophytes. Only a very small amount of chlorophyll a from green algae was found.
- Periphyton: Sixty-six periphyton taxa of eight different classes were identified in the two samples taken before exposure and differed from the sample taken on Day 29 after the start of exposure. The mean number of taxa and the mean diversity decreased over the course of the study. The total abundance increased from 213 000 cells/cm² to 781 000 cells/cm². However, the coefficient of variation for total abundance was very high. The periphyton was dominated by Cyanophyceae.
-Macrophytes: The following indigenous macrophyte species were found: Ceratophyllum demersum, Potamogeton natans and Zannichellia palustris. The macro-alga Chara globularis was also considered within the functional group of macrophytes. Macrophytes grew considerably during the exposure period, indicated by the increase of total area coverage of 16 % on Day -2 to 45 % on Day 29. On the last monitoring, Ceratophyllum demersum and Chara globularis covered 17 and 14 % of the area, respectively.

CLOEON DIPTERUM DETAILS
- Larvae: The abundance of Cloeon larvae before the start of the exposure was very similar in all enclosures. After one week of exposure (Day 6), mean larval abundance in the 10 μg a.s./L enclosures was significantly reduced to less than 50 % of control abundance, while no effects were found at the other treatment levels. After two weeks (Day 13), no larvae were found in the samples from the 10 μg a.s./L enclosures. At 3 μg a.s./L, abundance had decreased to 35 % of control abundance. At test concentrations up to and including 1 μg a.s./L, no effect was found. In weeks 3 and 4 (Day 20 and 27), abundance was significantly reduced at test concentrations 1 μg a.s./L and above, while abundance at 0.1 and 0.3 μg/L was still similar to controls. For detailed results on the abundance of Cloeon dipterum larvae see table 1 in “Any other information on results incl. tables”
- Emergence: Emergence traps were installed over one week before sampling. Emergence of Cloeon dipterum was not affected over the 5 weeks of exposure at concentrations up to and including 0.3 μg a.s./L, while at 10, 3 and 1 μg a.s./L, emergence dropped to zero in weeks 2, 3 and 4, respectively. In week 1 (Day 7) the MDD was slightly above 50 %, while later it was always < 50 %. In weeks 3 (Day 21) and 4 (Day 28), the MDD was < 30 %. Thus, the data allowed detection of ‘small effects’ (EFSA 2013) on emergence of Cloeon.
The NOEC for emergence of Cloeon dipterum was therefore 0.3 μg a.s./L while pronounced effects of unclear duration were found at concentrations of 1 μg a.s./L and higher. For detailed results on the Emergence of Cloeon dipterum [Ind./sampling] see table 2 in “Any other information on results incl. tables”.



Reported statistics and error estimates:
- Multiple t-tests of Williams (Williams, 1971, 1972). The Williams’ tests were performed one-sided with α = 0.05 (5% level of significance).

Table 1: Abundance of Cloeon dipterum larvae. Control geometric means, abundance related to abundance in the controls (%) per treatment level, NOEC (μg a.s./L) and related % MDD

Day after initial application

Geomean

% of control

NOEC

(μg a.s./L)

%MDD

Control

0.1 μg/L

0.3 μg/L

1.0 μg/L

3.0 μg/L

10 μg/L

-11

13.5

105

124

120

108

138

-

49

-1

27.6

104

93

118

102

104

-

44

6

11.9

100

118

134

89

42

3

45

13

10.3

90

89

104

34

0

1

47

20

8.3

62

73

29

0

0

0.3

37

27

5.4

116

137

37

0

0

0.3

44

34

1.6

47

167

73

14

0

3.0

87

  

Table 2: Emergence of Cloeon dipterum [Ind./sampling] Geometric means per treatment level, NOEC [μg a.s./L] and related % MDD and abundance related to abundance in the controls [%] per treatment level.

Day after initial application

Geomean

% of control

NOEC

(μg a.s./L)

%MDD

Control

0.1 μg/L

0.3 μg/L

1.0 μg/L

3.0 μg/L

10 μg/L

0

16.1

169

160

113

196

261

3

61

7

13.2

159

135

126

129

118

10

53

14

12.7

134

128

97

19

0

1

42

21

19.3

150

139

15

0

0

0.3

26

28

8.4

91

77

0

0

0

0.3

27

35

2.9

89

148

0

0

0

0.3

45

 

Validity criteria fulfilled:
yes
Conclusions:
The effects of chronic exposure to the test item on the mayfly, Cloeon dipterum, were assessed in outdoor mesocosms representative of a typical pond community (without fish or amphibians) during the summer. Through multiple dosings of the test item, time weighted average concentrations of thiamethoxam in the treated enclosures over the 5 weeks were maintained close to 100 % of nominal. Deviations of measured concentrations from the 80 to 120 % range were restricted to a few measurements. Thus, it is concluded that the effects can be related to the nominal concentrations. The variability between replicates was sufficiently low to detect small effects on larvae and emergence of adult mayflies in this mesocosm study at all but the final time point. Exposure to 0.1 and 0.3 μg a.s./L over five weeks had no effects on the abundance of larvae nor the emergence of Cloeon dipterum (NOEC = 0.3 μg a.s./L). Pronounced effects on both endpoints were found at 1 μg a.s./L.
Executive summary:

The effects of a chronic exposure to the test substance on the mayfly, Cloeon dipterum, were assessed in outdoor mesocosms representative of a typical pond community (without fish or amphibians) during the summer season.

Twenty stainless steel enclosures installed in a large artificial pond located at the test site were used for the test. The test systems contained indigenous flora and fauna (i.e., algae, macrophytes, zooplankton and macroinvertebrates) which originated from the sediments, water and aerial colonisation by flying insects. To test the effects of chronic exposure on mayflies, the following five nominal water concentrations were tested in three replicate enclosures while five enclosures served as untreated controls: 0.0 (control), 0.1, 0.3, 1.0, 3.0, 10 μg a.s./L (corresponding to 0.0 (control), 0.4, 1.2, 4.0, 12, 40 μg test item/L). Nine test item applications were conducted to achieve constant exposure over 7 weeks. The multiple dosings of the test item resulted in TWA concentrations between 93-108 % of the nominal test concentrations. Thus, effects can be related to the nominal concentrations. Variability of mayfly larval abundance and adult emergence between replicates was sufficiently low to detect small effects in such an outdoor mesocosm. The NOEC for effects on larval abundance and emergence of Cloeon dipterum was 0.3 μg a.s./L over the five weeks of constant exposure.

Description of key information

All available data were assessed and the studies representing the worst-case effects were included as key or weight-of-evidence studies. Other studies are included as supporting information. The key studies are considered to be worst-case and were selected for the CSA.

In the study that determines the effects of chronic exposure to the test substance on mayflies in an outdoor mesocosm, the NOEC for the most sensitive species, namely Cloeon dipterum, is determined to be 0.3 μg/L based on the effects on larval abundance and emergence. This 35-d NOEC is used as the key value for freshwater invertebrates (Hommen, 2016).

The 28-d NOEC of 0.56 mg a.i./L observed in saltwater species Americamysis bahia is used as the key value for saltwater invertebrates (Sayers, 2015).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
0.3 µg/L
EC10, LC10 or NOEC for marine water invertebrates:
0.56 mg/L

Additional information

Table: Overview of available data on the long-term toxicity to aquatic invertebrates

Species

Guideline / GLP

Endpoint

Effect value

Comment

Reference

Daphnia magna

(freshwater)

OECD 202, part II / GLP

21-d NOEC

100 mg/L

Semi-static test (3 weekly renewals). Measured concentrations well within ± 20% of nominal. Effect value is based on reproduction.

Neumann, 1997

Cloeon dipterum (freshwater)

OECD (2006) / GLP

35-d NOEC

0.3 µg a.i./L

Mesocosm study with special focus on the most sensitive species (mayfly; Cloeon dipterum) The 35-d NOEC of 0.3 µg/L is used in the aquatic hazard evaluation as the most sensitive aquatic invertebrate value.

Hommen, 2016

Americamysis bahia

(saltwater)

CSPP Draft Guideline 850.1350

28-d NOEC

0.56 mg/L

Flow through regime. Measured concentrations well within ± 20% of nominal. Effect value is based on survival

Sayers, 2015