bis(branched-alkyl) 2-{[(1-phenylethyl)amino]methyl}alkanedioate

Sediment toxicity

Link to relevant study record(s)

Reference

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 October 2017 to 07 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 233 (Sediment-Water Chironomid Life-Cycle Toxicity Test Using Spiked Water or Spiked Sediment)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

ANALYTICAL SAMPLING
- Stocks used to dose the F0 generation sediments were analyzed as soon as possible after preparation to confirm the dosing concentrations. During the first and second generation, one of the additional replicate test chambers prepared for each treatment and control group was collected for analysis of overlying water, pore water and sediment on each of Days 0, 14 and 28, and were processed immediately for analysis. Back-up samples of overlying water were also collected and stored but were not needed during the test. Additionally, samples of overlying water pooled from the crystallizing dishes in each experimental group, as well as sediment from each crystallizing dish, were collected at the end of the first generation emergence (Day 28).

Vehicle:

yes

Remarks:

acetone

Details on sediment and application:

TEST SEDIMENT
- Formulated sediment based on the recommendations of OECD Guideline 233 (1) was used as the test sediment. The sediment was composed of approximately 5% sphagnum peat moss, 20% silt and clay (kaolin clay) and 75% industrial quartz sand. The amount of peat moss added to the mixture was adjusted for the percent moisture content of 52.3%. The dry constituents of the sediment were mixed together and a sample of the formulated sediment used in the test was sent to Agvise Laboratories, Northwood, North Dakota, for characterization and analysis of total organic carbon (TOC) and a summary of sediment characterization was presented in the full study report. The percent organic carbon of the sediment was determined to be 1.2%. The pH of the sediment was 7.6. The results of periodic analyses performed to measure the concentrations of selected organic and inorganic constituents in a representative sample of formulated sediment like that used in the test were provided in the full study report.

PREPARATION OF TEST CONCENTRATIONS
- The test substance was administered to the test organism in sediment. This route of administration was selected because it represents the most likely route of exposure to sediment dwelling organisms.
- Individual dosing stock solutions were prepared for use in spiking the sediment to prepare each of the five concentrations tested. Test concentrations were not adjusted for the active ingredient of the test substance during preparation, and are based on the test substance as received. A primary stock solution of non-radiolabeled test substance was prepared by mixing 5.25 g of test substance into HPLC-grade acetone at a nominal concentration of 105 mg/mL. The radiolabeled test substance was received as a stock with a nominal concentration of 11.5 mg/mL. The radioactivity of the stock was confirmed prior to use in the study by analyzing three, 100-μL samples of stock solution by liquid scintillation counting (LSC). The measured concentration of the primary radiolabeled stock was 10.9 mg/mL, or 95.0% of the expected radioactivity.
- The dosing stock solutions were prepared as isotopic dilutions at nominal concentrations of 6.3, 12.5, 25, 50 and 100 mg/mL. A 0.061-mL aliquot of the radiolabeled primary stock (equivalent to 0.665 mg of test substance) was added to five 25-mL volumetric flasks. The remaining balance of test substance needed to produce the nominal concentrations was achieved with the addition of calculated volumes of non-radiolabeled primary stock solution. The flasks were brought to volume with acetone and the solutions were inverted to mix. The stocks ranged in appearance from clear and colorless to clear and slightly yellow with color intensity decreasing with decreasing concentration between the 100 and 50 mg/mL stocks with no visible precipitates after mixing.
- To prepare a batch of sediment for each treatment level, a 23-mL volume of the appropriate stock solution was added to 115.0 grams of sand in a labeled glass beaker and was stirred with a glass stir rod until homogenous. This dosed “sand premix” was placed under a fume hood and the acetone was allowed to evaporate for approximately one and a half hours. The 115-gram sand premix was added to 1185 g dry weight of untreated formulated sediment (1262 g wet weight) in a 2000 mL plastic Nalgene bottle and mixed on a rotary mixer for approximately one hour. Additional formulated sediment, 1000 g dry weight (1065 g wet weight) was added to the premix to achieve a final weight of 2300 grams. This 2300 g batch of sediment was mixed on a rotary mixer for approximately 16.5 hours prior to transfer of the sediment to the test chambers. Since a solvent (acetone) was used in the preparation of the test sediments, a solvent control was included in the test design. The solvent control sediment was prepared using 23 mL of acetone, with the same mixing procedures as the treated sediments but with no test substance added. The negative control sediment was prepared without the addition of test substance or solvent.
- Dosed sediments were prepared three times for the study; once prior to the start of the first generation, once prior to the use of crystallizing dishes and once prior to the start of the second generation. All three preparations followed the dosing procedure as described above; however, the final weights of the sediment batches differed based on the amount of sediment needed for the test chambers or crystallizing dishes. The amount of test substance added was proportional to the amount of sediment being dosed. The ratio of radiolabeled vs. non-radiolabeled material remained constant. The percent moisture of the sediment was determined prior to use.

PREPARATION OF TEST CHAMBERS
- Twelve replicate test chambers were prepared for each treatment and control group for both the first and second generations. Eight replicates per group were used for biological observations. An additional three replicates per group were prepared for use, as needed, in analytical confirmation of concentrations on Days 0, 14 and 28. An additional replicate was included as an extra analytical sample, if needed. After mixing the batch sediments, approximately 2 cm (approximately 150 mL) of the appropriate dosed formulated sediment was placed in the bottom of each test chamber (glass jars) on a top-loading balance, and the weight of the sediment was recorded. Approximately 600 mL of overlying water was slowly added to each test chamber, while avoiding disturbance of the sediment, and each test chamber was loosely covered. After preparation, the test chambers were indiscriminately arranged in a temperature-controlled environmental chamber, and gentle aeration was applied to each test chamber. The sediment/water mixtures were allowed to acclimate under static conditions for approximately 48 hours prior to introduction of the organisms.
- On Day 10 of the test, newly prepared dosed sediment and clean overlying water was added to crystallizing dishes used for the first generation reproduction so that the dishes could equilibrate for 48 hours before the first egg ropes were produced. Two replicate crystallizing dishes were prepared for each experimental group. The dishes were prepared with an approximate 1 : 4 ratio of sediment depth to overlying water depth. Approximately 527 grams of appropriate sediment and 1200 mL of UV sterilized well water were added to each crystallizing dish.

TEST APPARATUS
- The test chambers for the first and second generation larval exposures were 1 quart (approximately 950-mL) glass jars containing approximately 2 cm (approximately 150 mL) of sediment and 600 mL of overlying water. The depth of the sediment measured in a representative chamber was 2.0 cm, and the depth of the overlying water in a representative chamber was 7.0 – 8.0 cm. The approximate 1:4 ratio of sediment depth to overlying water depth was maintained throughout the test by periodically replacing water lost due to evaporation and sampling for water quality measurements, with reverse-osmosis water. Loose plastic covers were placed over each test chamber during the test, and aeration was applied to each test chamber through a glass pipette that extended to a depth not closer than 2 cm to the surface of the sediment. Air was bubbled into the test chambers at a rate that was greater than one bubble per second, but not so great as to disturb the sediment. The test chambers were indiscriminately arranged in a temperature-controlled environmental chamber during the test, and were labeled with the project number, test concentration and replicate designation. The general operation of the test apparatus was checked visually at least once each day during the test.
- Breeding cages for the first generation adult midges consisted of Plexiglass boxes measuring 30 cm in all three dimensions, with gauze covering an opening on the top and on one side. Each cage contained a 2 L crystallizing dish containing sediment and overlying water in an approximate 1 : 4 ratio of sediment depth to overlying water depth. Egg ropes collected from the crystallizing dishes were placed into a well plate (12 Well Culture Cluster) assigned to each breeding cage, with each well containing at least 2.5 mL of water from the corresponding crystallizing dish. Each well plate was covered with a lid.

Test organisms (species):

Chironomus riparius

Details on test organisms:

TEST ORGANISM
- The midge, Chironomus riparius, was selected as the test species for this study. This species is representative of an important group of aquatic invertebrates and was selected for use in the study based upon past history of use in the laboratory and the recommendation of the study guideline. Midges used in the test were obtained as egg masses from cultures maintained by EAG Laboratories, Easton, Maryland. The identity of the species was verified by the supplier of the original culture, Aquatic Research Organisms, Hampton, New Hampshire.
- Larvae used to start the test were collected from 10 separate egg masses and were hatched in water from the same source and at approximately the same temperature as was used during the test. At the time of test initiation, the larvae were 1 to 3 days old. During the holding period, water temperatures ranged from 19.3 to 21.1 °C, the pH of the water ranged from 8.0 to 8.6, and the dissolved oxygen concentrations were ≥8.0 mg/L (≥88% of saturation). The larvae showed no signs of disease or stress prior to test initiation.

FEEDING
- During the holding period, the midge larvae were fed a suspension of TetraMin flake food daily. During the test, the midges were fed ground TetraMin flake food approximately three times per week beginning on Day 0, excluding the additional abiotic test chambers prepared for analytical sampling.
- Individual aliquots of approximately 10 - 30 mg of the ground food were supplied to each test chamber at each feeding. The results of periodic analyses performed to measure the concentrations of selected organic and inorganic constituents in a representative sample of TetraMin flake food

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

28 d

Exposure phase:

larvae from first generation (P)

Duration:

28 d

Exposure phase:

larvae from second generation (F1)

Post exposure observation period:

Not applicable

Hardness:

- First generation (hardness): 152 to 204 mg/L as CaCO3
- Crystalizing dishes (hardness): 208 to 292 mg/L as CaCO3
- Second generation (hardness): 164 to 180 mg/L as CaCO3
- First generation (alkalinity): 136 to 174 mg/L as CaCO3
- Crystalizing dishes (alkalinity): 178 to 228 mg/L as CaCO3
- Second generation (alkalinity): 110 to 178 mg/L as CaCO3

Test temperature:

- First generation: 19.1 to 20.2 °C
- Crystalizing dishes: 18.6 to 19.1 °C
- Second generation: 19.3 to 20.1 °C

pH:

- First generation: 8.4 to 9.3
- Crystalizing dishes: not measured
- Second generation: 8.3 to 9,6

Dissolved oxygen:

- First generation: ≥ 86%; aerated
- Crystalizing dishes: not measured
- Second generation: ≥ 73%; aerated

Salinity:

Not applicable

Ammonia:

- First generation: < LOQ to 0.211 mg/L as NH3
- Crystalizing dishes: < LOQ to 0.373 mg/L as NH3
- Second generation: < LOQ mg/L as NH3
- The limit of quantitation (LOQ) for measurements of ammonia was 0.17 mg/L as NH3 based on the lowest concentration standard used to calibrate the meter used for measurement.

Conductivity:

- First generation: 385 to 498 μS/cm
- Crystalizing dishes: 515 to 636 μS/cm
- Second generation: 342 to 461 μS/cm

Nominal and measured concentrations:

- Negative control: 0 mg/kg sediment dw (nominal); < LOQ (measured mean)
- Solvent control: 0 mg/kg sediment dw (nominal); < LOQ (measured mean)
- Test item 63 mg/kg sediment dw (nominal); 53 mg/kg sediment dw (measured mean)
- Test item 125 mg/kg sediment dw (nominal); 108 mg/kg sediment dw (measured mean)
- Test item 250 mg/kg sediment dw (nominal); 205 mg/kg sediment dw (measured mean)
- Test item 500 mg/kg sediment dw (nominal); 432 mg/kg sediment dw (measured mean)
- Test item 1000 mg/kg sediment dw (nominal); 827 mg/kg sediment dw (measured mean)
- Mean measured concentration calculated as the mean of the measured concentrations on Day 0 of the F0 and F1 generations.

Details on test conditions:

OBJECTIVE
- The objective of this study was to determine the effects of sediment-incorporated test item on two generations of the midge, Chironomus riparius, during a 28-day exposure period under static test conditions for each generation. The evaluated endpoints for both the first and second generations were the total number of adults emerged, development rate, and sex ratio of fully emerged and alive adults. The number of egg ropes per female and the fertility of the egg ropes was also evaluated for the first generation.

EXPERIMENTAL DESIGN
- Midges were exposed to a geometric series of five test concentrations, a negative control (untreated formulated sediment), and a solvent control (acetone) for 28 days under static test conditions for each of the two generations. Exposure in each generation consisted of eight replicate test chambers maintained in each treatment and control group, with 20 midges in each test chamber, for a total of 160 midges per test concentration, per generation. Each test chamber contained sediment and overlying water. An additional three replicates were prepared in each treatment and control group for use in analytical sampling (for each generation). No midges were placed in the additional replicates sampled on Day 0, but those sampled on Days 14 and 28 had midges added at the same time as the “biological” replicates on Day 0. These additional replicates were not used to evaluate the biological response of the test organisms.
- Test concentrations in the sediment were prepared on a mg/kg dry weight basis. Nominal test concentrations were selected in consultation with the Sponsor based on exploratory range-finding toxicity data, and were 63, 125, 250, 500 and 1000 mg test item/kg of sediment. Test concentrations were measured in samples of overlying water, pore water, and sediment collected on Day 0, Day 14, and on Day 28 of each generation. Additionally, samples of overlying water pooled from the crystallizing dishes in each experimental group as well as sediment and pore water from each crystallizing dish were collected at the end of the first generation exposure. The results of the study are based on mean measured test concentrations in the sediment measured on Day 0 for both generations.
- The water/sediment systems in the test compartments were allowed to equilibrate for approximately 48 hours prior to introduction of the organisms. First-instar, 1 to 3-day old midge larvae were impartially assigned to exposure chambers at test initiation. Observations of abnormal behavior were made daily throughout the test. In the first generation, chironomid emergence, time to emergence and sex ratio of the fully emerged and alive midges was assessed. Emerged adults were transferred to breeding cages (four replicates assigned to each of two cages, per experimental group) to facilitate swarming, mating and oviposition. The number of egg ropes produced and their fertility was assessed. From these egg ropes, first instar larvae were placed into freshly prepared test chambers to determine the viability of the second generation through an assessment of their emergence, time to emergence and the sex ratio of fully emerged and alive midges. Observations of the effects of the test item on the total number of adults emerged, development rate, and sex ratio of fully emerged and alive adults in both the first and second generations, as well as the number of egg ropes per female and fertility of the egg ropes from the first generation were used to determine the no-observed effect concentration (NOEC) and the lowest-observed effect concentration (LOEC).

TEST WATER
- The water used for holding and testing was freshwater obtained from a well approximately 40 meters deep located on the EAG Laboratories-Easton site. The well water was passed through a sand filter to remove particles greater than approximately 25 µm, and pumped into a 37,800 L storage tank where the water was aerated with spray nozzles. Prior to use in the test system, the water was filtered to 0.45 µm to remove fine particles and was passed through an ultraviolet (UV) sterilizer.
- The well water is characterized as moderately-hard water. The specific conductance, hardness, alkalinity, pH and total organic carbon (TOC) content of the well water during the approximate four week period immediately preceding the test were included in the full study report. The results of periodic analyses performed to measure the concentrations of selected organic and inorganic constituents in the well water were included in the full study report.

ENVIRONMENTAL CONDITIONS
- The test systems were illuminated using fluorescent tubes that emit wavelengths similar to natural sunlight. The lights were controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 30-minute transition period of low light intensity was provided when lights went on and off to avoid sudden changes in light intensity. Light intensity was measured at the water surface of one representative test chamber at the beginning of each phase using a SPER Scientific Model 840006 light meter.
- The test was conducted at a target water temperature of 20 ± 2 C. Temperature was measured in the overlying water of one replicate test chamber of each treatment and control group daily during the test using a digital thermometer. Measurements typically rotated among replicate test chambers in each group at each measurement interval. Temperature was also measured in all replicate test chambers at the end of the emergence period in each generation (Day 28 of exposure), and in the overlying water in the crystallizing dishes in breeding cages for the controls and highest treatment group (1000 mg/kg) at the beginning and end of the reproductive period. Water temperature also was monitored continuously in a container of water placed adjacent to the test chambers using a validated environmental monitoring system (AmegaView Central Monitoring System). The system measurements were verified prior to exposure initiation with a digital thermometer.
- Dissolved oxygen concentrations were measured in samples of overlying water from one replicate test chamber of each treatment and control group daily during the test using a Thermo Scientific Orion Star A213 Benchtop RDO/DO meter. Measurements of pH were made in samples of overlying water from one replicate test chamber of each treatment and control group at test initiation, once each week during the test, and at test termination using a Thermo Scientific Orion Dual Star pH/ISE meter. Measurements of dissolved oxygen and pH typically rotated among replicate test chambers in each group at each measurement interval. Additionally, dissolved oxygen and pH were measured in the overlying water of all replicate test chambers at the end of the emergence period in each generation (Day 28 of exposure).
- Hardness, alkalinity, specific conductance and ammonia were measured in composite samples of overlying water from the negative and solvent control group replicates and the highest concentration treatment group replicates at the beginning and end of the first and second generation exposures. Hardness and alkalinity measurements were made by titration based on methods in Standard Methods for the Examination of Water and WastewateR. Specific conductance was measured using an Thermo Scientific Orion Star A122 Portable Conductivity meter. Ammonia was measured using a Thermo Scientific Orion Dual Star pH/ISE meter. Additionally, hardness, alkalinity, specific conductance and ammonia were also measured in composite samples of overlying water from the crystallizing dishes in the negative control, solvent control and highest concentration treatment group at the beginning and end of the reproductive exposure.

Reference substance (positive control):

no

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

108 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

fertility

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

53 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

fertility

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

> 827 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: emergence ratio

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

827 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: emergence ratio

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

> 827 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

development time

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

827 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

development time

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

108 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

development rate

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

53 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

development rate

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

> 827 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

sex ratio

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

827 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

sex ratio

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

> 827 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

fecundity

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

827 mg/kg sediment dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

fecundity

Details on results:

MEASUREMENT OF TEST CONCENTRATIONS
- Nominal concentrations selected for use in this study were 63, 125, 250, 500 and 1000 mg/kg dry weight of sediment. During the course of the test, the appearance of the overlying water was observed in the test chambers. At test initiation for the first and second generations, the overlying water appeared clear and colorless. At test termination, the overlying water appeared clear and yellow.
- Results of LSC analyses to measure the concentration of the test item in the primary radiolabelled stock solution are presented in Table 1 (attached). The primary 14C-labeled stock had measured concentrations of 10.8, 11.0 and 11.0 mg/mL, corresponding to 93.8, 95.4 and 95.8% of nominal. Measured concentrations of the test item in the working stock solutions used to dose the sediment are presented in Table 2 (attached). The working stocks had measured concentrations of 6.0, 11.9, 24.3, 48.2 and 95.5 mg/mL, corresponding to 94.8, 95.5, 97.2, 96.4 and 95.5% of nominal.
- Results of LSC analyses to measure concentrations of the test item equivalents in the sediment, overlying water and pore water samples during the test are presented in Tables 3, 4 and 5 (attached), respectively for the first generation and Tables 6, 7 and 8 (attached), respectively for the second generation. Results of LSC analyses to measure concentrations of the test item equivalents in the overlying water in the crystalizing dishes are presented in Table 9 (attached). All reported sediment concentrations are expressed on a dry weight basis with an LOQ of 0.273 mg/kg dry sediment. The LOQ for overlying and pore water analyses was 0.00630 mg/L. Measured concentrations of the test item equivalents in negative and solvent control sediment and water samples on Days 0, 14 and 28 in the first and second generations were below the LOQ. Measured concentrations in the sediment samples collected from the treatment groups from both generations ranged from approximately 68.0 to 104% of nominal. Measured concentrations in the overlying water and pore water samples from both generations ranged from approximately 0.200 to 16.0 mg/L and 1.23 to 45.0 mg/L, respectively. When measured concentrations of the sediment samples collected during both generations of the test were averaged, the mean measured test concentrations for this study were 50, 99, 197, 408 and 832 mg/kg, representing 78.5, 79.4, 78.8, 81.5 and 83.2% of nominal concentrations, respectively. The results of the study were based on the mean measured concentrations in sediment. The concentrations measured in the sediment in the crystallizing dishes were not used in the calculation of mean measured sediment concentrations, since the organisms were not directly exposed to the sediment at that time. Instead the organisms were allowed to swarm in the breeding cages above the spiked sediment.

OBSERVATIONS AND MEASUREMENTS
- Measurements of temperature, dissolved oxygen and pH of the overlying water in the test chambers are summarized in Tables 10, 11 and 12 (attached). All water quality measurements were within the desired ranges. Water temperatures were within the 20 ± 2 °C range established for the test. Measurements of pH ranged from 8.3 to 9.6 during the test. Dissolved oxygen concentrations remained ≥6.6 mg/L (≥73% of saturation) throughout the test. Measurements of specific conductance, hardness, alkalinity and ammonia of the overlying water in the negative control and the highest concentration treatment group are summarized in Tables 10, 11 and 12 (attached). Measurements of specific conductance in the first generation and the crystallizing dishes were slightly higher than typical water quality of EAG Laboratories-Easton well water, but the second generation specific conductance was closer to typical measurements. Hardness and alkalinity were higher and lower (respectively) than typical water quality of EAG Laboratories-Easton well water during the first and second generations and higher than typical in the overlying water of the crystallizing dishes. The departure from typical measurements did not have a negative impact on the results of the test as is demonstrated by the control groups meeting the validity criteria established for the test by the OECD 233 guideline. Measurements of ammonia in the overlying water during the first and second generations and in the crystallizing dishes ranged from below the limit of quantitation (- No unusual observations of organisms avoiding the sediment occurred during the test. There were a few observations of larvae on the surface of the sediment or swimming in the water column during the study, but these did not appear to be dose-responsive, were comparable between the control and treatment groups, and were not considered to be treatment-related. There also were a few observations of dead pupae or larvae and adults that emerged and died. However, these numbers were small, were not concentration responsive, and were not considered to be treatment-related.
- There were no apparent treatment-related effects on emergence at any concentration tested. The numbers of emerged midges and emergence ratios in each control and treatment group are summarized in Tables 13 and 14 (attached). During the first generation emergence was first noted on Day 12 of the test and continued through Day 24. The mean emergence ratios in the negative control, solvent control and the 53, 108, 205, 432 and 827 mg/kg treatment groups were 0.97, 0.92, 0.94, 0.91, 0.93, 0.94 and 0.88, respectively. During the second generation, emergence was first noted on Day 13 of the exposure and continued through Day 28. The mean emergence ratios in the negative control, solvent control, pooled control and the 53, 108, 205, 432 and 827 mg/kg treatment groups were 0.91, 0.91, 0.91, 0.96, 0.99, 0.90, 0.96 and 0.82, respectively. There were no significant differences in comparisons of emergence ratios between the negative control and the treatment groups or the solvent control and treatment groups using a Dunnett’s test (p > 0.05) for the first generation. There was a significant difference in comparisons of emergence ratios between the 0.827 mg/kg treatment group and the 108 and 432 mg/kg treatment groups using a Kruskal-Wallis test (p ≤ 0.05) for the second generation; however, the difference was not considered to be biologically meaningful as there was no difference observed from the pooled control or the solvent control. Therefore, the NOEC for emergence ratio was 827 mg/kg, the highest concentration tested. The LOEC for emergence ratio was determined to be >827 mg/kg.
- During the first generation, the mean development time in the negative control, solvent control, pooled control, 53, 108, 205, 432 and 827 mg/kg treatment groups was 15.6, 15.1, 15.3, 15.7, 15.2, 15.8, 16.4 and 16.5 days, respectively (see Table 13, attached). Since there were no statistical difference between the negative and solvent control for the first generation development time data, the treatment groups were compared to the pooled control and the solvent control. There was a statistical difference between the negative and solvent control for the second generation development time data; therefore, the treatment groups were compared to the negative control and solvent control separately. There were no statistically significant differences between the pooled control or the solvent control and the treatment group first generation development time data using a Bonferroni t-test (pooled control) or a Dunnett’s test (solvent control). During the second generation, the mean development time in the negative control, solvent control, 53, 108, 205, 432 and 827 mg/kg treatment groups was 15.9, 15.0, 15.3, 16.1, 15.8, 16.3 and 16.4 days, respectively (see Table 14, attached). There were no statistical differences between the negative control or solvent control and any of the treatment groups for the second generation development time data. Therefore, the NOEC for development time was 827 mg/kg, the highest concentration tested. The LOEC was >827 mg/kg.
- Since there were no significant interactions between sex and treatment for the first generation development rate, the sexes were pooled for analysis. There were no statistical differences between the negative and solvent control development rates for the first generation data; therefore the treatment groups were compared to the pooled control and additionally, to the solvent control data. During the first generation, mean development rates (defined as the portion of larval development which takes place per day) in the negative control, solvent control, pooled control, 53, 108, 205, 432 and 827 mg/kg treatment groups were 0.0671, 0.0695, 0.0683, 0.0667, 0.0685, 0.0663, 0.0636 and 0.0634. There were statistically significant differences (p < 0.05) in mean development rate for the 432 and 827 mg/kg treatment groups during the first generation when compared to the pooled control using a Bonferroni t-test. There were also statistically significant differences (p < 0.05) in mean development rate for the 205, 432 and 827 mg/kg treatment groups during the first generation when compared to the solvent control using a Dunnett’s test. There was a statistical interaction between the treatments and sex for the second generation development rate data; therefore the development rate data for each sex was analyzed separately. The mean development rates in each control and treatment group are summarized in Tables 13 and 14 (attached). The mean male development rates for the second generation organisms were 0.0691, 0.0742, 0.0735, 0.0706, 0.0683, 0.0677 and 0.0678, respectively for the negative control, solvent control, 53, 108, 205, 432 and 827 mg/kg treatment groups. The mean female development rates for the second generation organisms were 0.0621, 0.0647, 0.0636, 0.0600, 0.0630, 0.0610 and 0.0607, respectively for the negative control, solvent control, 53, 108, 205, 432 and 827 mg/kg treatment groups.
- There was a significant difference between the negative control and solvent control data for both sexes; therefore, the data for each sex was compared to the negative control and solvent control data separately. There were no significant differences in comparison of the second generation development rates between the negative control and the treatment groups using Dunnett’s test (p > 0.05) for either sex. There were statistically significant differences in the second generation male development rate in the 108, 205, 432 and 827 mg/kg treatment groups when compared to the solvent control using a Dunnett’s test. There were also statistically significant differences in the second generation female development rates in the 108, 432 and 827 mg/kg treatment groups when compared to the solvent control using a Dunnett’s test. Therefore, the NOEC for development rate was 53 mg/kg and the LOEC was 108 mg/kg, based on the significance observed in the second generation data.
- The sex ratio was determined for each experimental group for each generation. In the first generation, the male sex ratio for the negative control, solvent control, 53, 108, 205, 432 and 827 mg/kg treatment groups was 0.40, 0.46, 0.37, 0.41, 0.46, 0.44 and 0.47, respectively. The female sex ratio was 0.57, 0.46, 0.57, 0.49, 0.47, 0.49 and 0.41, respectively (see Table 15, attached). In the second generation, the male sex ratio for the negative control, solvent control, 53, 108, 205, 432 and 827 mg/kg treatment groups was 0.44, 0.48, 0.44, 0.45, 0.49, 0.47 and 0.38, respectively. The female sex ratio was 0.48, 0.44, 0.51, 0.54, 0.41, 0.49 and 0.44, respectively. The sex data was analyzed as the number of males that emerged in a treatment group compared to the total number of emerged adults in the group, using a Bonferroni t-test. No significant differences were observed between the pooled control and the treatment groups. Therefore, the NOEC and LOEC for sex ratio are 827 and >827 mg/kg, respectively.
- The fecundity and fertility of the egg ropes produced by the first generation midges was also analyzed. A summary of the fecundity and fertility of the egg ropes produced during the first generation is presented in Table 16 (attached). There was a statistical difference in fecundity in the 205 mg/kg treatment group using a Jonckheere Terpstra trend test; however, the difference was not dose responsive and was therefore not considered to be treatment related. There were statistical differences in fertility in the 108, 205, 432 and 827 mg/kg treatment groups using a Jonckheer-Terpstra trend test. Therefore, the NOEC and LOEC for fertility of the egg ropes (most sensitive of the two endpoints) are 53 and 108 mg/kg, respectively. The NOEC and LOEC for fecundity of the egg ropes was 827 and >827 mg/kg, respectively.

CONDITIONS FOR VALIDITY OF THE TEST
-The following criteria were used to judge the validity of the test and were met in this study with
one exception:
1. There was >70% emergence at the end of the exposure period for both generations in the control groups.In the first generation, emergence in the negative and solvent controlgroups was 97 and 92%, respectively. In the second generation, emergence in thenegative and solvent control groups was 91 and 91%.
2. Greater than 85% of the total emerged adults from the controls occurred between 12 and 23 days after the insertion of the first instar larvae into the test chambers. In this test, 100% of the total emerged adults occurred between 12 and 23 days after insertion into the test chambers for both generations.
3. The mean sex ratio of fully emerged and alive adults (as female or male fraction) in the controls of both generations was between 0.4 and 0.6.
4. For each breeding cage, the number of egg ropes in the controls of the first generation was greater than 0.6 per female added to the breeding cage.
5. The fraction of fertile egg ropes in each breeding cage of the controls of the first generation were not greater than 0.6. Breeding cage one and two for the negative control had fractions of 0.57 and 0.59 fertile egg ropes per breeding cage, respectively. The solvent control had 0.53 and 0.43 fertile egg ropes per cage, respectively.
6. The pH of the overlying water in all test chambers at the end of the test were between 6 and 9 and the dissolved oxygen concentration was greater than 60% of the air saturation value. The pH at the end of the test ranged from 8.0 to 8.9 and the dissolved oxygen concentration was ≥71%.
8. The water temperature did not differ by more than ± 1 °C. Temperatures measured in the overlying water of the test chambers throughout the test ranged from 19.1 to 20.2 °C.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

See below

STATISTICAL ANALYSIS

- Data from the negative and solvent control groups for each parameter were compared using a t-test. Since a significant difference was detected between the two control groups for the first generation emergence ratio and the second generation development time and development rate (p≤0.05), the data from the treatment groups were compared to the negative control data and the solvent control data separately to evaluate treatment-related differences. Since there were no significant differences detected between the two control groups for the first generation development time and development rate and the second generation emergence ratio, the data from the treatment groups were able to be compared to the pooled control group, and were additionally compared to the solvent control group. Data from all endpoints were evaluated for normality (Chi-Square or Shapiro-Wilk’s) and homogeneity of variance (Bartlett’s or Levene’s ) (p = 0.01). Since the first generation development time, development rate, and emergence ratio data as well as the second generation development time and development rate data passed the assumptions of normality and homogeneity, the data in the treatment groups were compared to the negative and solvent control data using a Dunnett’s test or to the pooled control using a Bonferroni t-test to identify any significant differences (p = 0.05). The statistical tests were conducted using a personal computer with TOXSTAT or SAS software. Since the assumption of normality was not met in the second generation emergence ratio data, an attempt was made to correct the condition by arc sine square root transformation of the data. Since the transformation did not correct the problem, the data in the treatment groups were compared to the pooled control data using a Kruskal-Wallis non parametric test to identify any significant differences (p = 0.05). The statistical tests were conducted using a personal computer with TOXSTAT. The assumption of homogeneity was not met for the fertility and fecundity data. The data in the treatment groups were compared to the pooled control data using a Jonckheere-Terpstra trend test to identify any significant differences (p = 0.05). The statistical tests were conducted using a personal computer with SAS software.

- For each generation, a preliminary analysis was performed to determine if there were differences in the sensitivity of sexes to the test substance. The preliminary analysis consisted of two tests: a test for concentration-related trend in the proportion of emerged males and females in each treatment group, and a test of the significance of the interaction between sex and treatment in an ANOVA performed on development rate. Since neither of the two tests for the first generation development rate were significant at the 0.05 probability level, the sexes were pooled in subsequent analyses. There was a significant difference (p≤0.05) between the sexes for the second generation development rate; therefore, the sexes were analysed separately.

Validity criteria fulfilled:

yes

Conclusions:

Two generations of the midge (Chironomus riparius) were exposed for 28 days to five mean measured concentrations of sediment-incorporated test item ranging from 53 to 827 mg/kg. There were treatment-related effects observed on fertility of the egg ropes in the 108, 205, 432 and 827 mg/kg treatment groups. Fertility and development rate were the most sensitive endpoints. Based on the mean measured concentrations in sediment, the NOEC was 53 mg/kg and the LOEC was 108 mg/kg. The NOEC and LOEC for emergence ratio, development time, sex ratios and fecundity were 827 and >827 mg/kg, respectively.

Executive summary:

GUIDELINE

The study was based on procedures in the OECD Guidelines for the Testing of Chemicals, Guideline 233:Sediment-Water Chironomid Life-Cycle Toxicity Test Using Spiked Water or Spiked Sedimentand ASTM Standard E 1706-05:Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates.

 

METHODS

The objective of this study was to determine the effects of sediment-incorporated test item on two generations of the midge,Chironomus riparius, during a 28-day exposure period under static test conditions for each generation. The evaluated endpoints for both the first and second generations were the total number of adults emerged, development rate, and sex ratio of fully emerged and alive adults. The number of egg ropes per female and the fertility of the egg ropes was also evaluated for the first generation.

 

Midges were exposed to a geometric series of five test concentrations, a negative control (untreated formulated sediment), and a solvent control (acetone) for 28 days under static test conditions for each of the two generations. Exposure in each generation consisted of eight replicate test chambers maintained in each treatment and control group, with 20 midges in each test chamber, for a total of 160 midges per test concentration, per generation. Each test chamber contained sediment and overlying water. An additional three replicates were prepared in each treatment and control group for use in analytical sampling (for each generation). No midges were placed in the additional replicates sampled on Day 0, but those sampled on Days 14 and 28 had midges added at the same time as the“biological”replicates on Day 0. These additional replicates were not used to evaluate the biological response of the test organisms.

 

Test concentrations in the sediment were prepared on a mg/kg dry weight basis. Nominal test concentrations were selected in consultation with the Sponsor based on exploratory range-finding toxicity data, and were 63, 125, 250, 500 and 1000 mg test item/kg of sediment. Test concentrations were measured in samples of overlying water, pore water, and sediment collected on Day 0, Day 14, and on Day 28 of each generation. Additionally, samples of overlying water pooled from the crystallizing dishes in each experimental group as well as sediment and pore water from each crystallizing dish were collected at the end of the first generation exposure. The results of the study are based on mean measured test concentrations in the sediment measured on Day 0 for both generations.

 

The water/sediment systems in the test compartments were allowed to equilibrate for approximately 48 hours prior to introduction of the organisms. First-instar, 1 to 3-day old midge larvae were impartially assigned to exposure chambers at test initiation. Observations of abnormal behavior were made daily throughout the test. In the first generation, chironomid emergence, time to emergence and sex ratio of the fully emerged and alive midges was assessed. Emerged adults were transferred to breeding cages (four replicates assigned to each of two cages, per experimental group) to facilitate swarming, mating and oviposition. The number of egg ropes produced and their fertility was assessed. From these egg ropes, first instar larvae were placed into freshly prepared test chambers to determine the viability of the second generation through an assessment of their emergence, time to emergence and the sex ratio of fully emerged and alive midges. Observations of the effects of the test item on the total number of adults emerged, development rate, and sex ratio of fully emerged and alive adults in both the first and second generations, as well as the number of egg ropes per female and fertility of the egg ropes from the first generation were used to determine the no-observed effect concentration (NOEC) and the lowest-observed effect concentration (LOEC).

 

RESULTS

No unusual observations of organisms avoiding the sediment occurred during the test. There were a few observations of larvae on the surface of the sediment or swimming in the water column during the study, but these did not appear to be dose-responsive, were comparable between the control and treatment groups, and were not considered to be treatment-related. There also were a few observations of dead pupae or larvae and adults that emerged and died. However, these numbers were small, were not concentration responsive, and were not considered to be treatment-related.

 

There were no apparent treatment-related effects on emergence at any concentration tested. During the first generation emergence was first noted on Day 12 of the test and continued through Day 24. The mean emergence ratios in the negative control, solvent control and the 53, 108, 205, 432 and 827 mg/kg treatment groups were 0.97, 0.92, 0.94, 0.91, 0.93, 0.94 and 0.88, respectively. During the second generation, emergence was first noted on Day 13 of the exposure and continued through Day 28. The mean emergence ratios in the negative control, solvent control, pooled control and the 53, 108, 205, 432 and 827 mg/kg treatment groups were 0.91, 0.91, 0.91, 0.96, 0.99, 0.90, 0.96 and 0.82, respectively. There were no significant differences in comparisons of emergence ratios between the negative control and the treatment groups or the solvent control and treatment groups using a Dunnett’s test (p > 0.05) for the first generation. There was a significant difference in comparisons of emergence ratios between the 0.827 mg/kg treatment group and the 108 and 432 mg/kg treatment groups using a Kruskal-Wallis test (p≤0.05) for the second generation; however, the difference was not considered to be biologically meaningful as there was no difference observed from the pooled control or the solvent control. Therefore, the NOEC for emergence ratio was 827 mg/kg, the highest concentration tested. The LOEC for emergence ratio was determined to be >827 mg/kg.

 

During the first generation, the mean development time in the negative control, solvent control, pooled control, 53, 108, 205, 432 and 827 mg/kg treatment groups was 15.6, 15.1, 15.3, 15.7, 15.2, 15.8, 16.4 and 16.5 days, respectively. Since there were no statistical difference between the negative and solvent control for the first generation development time data, the treatment groups were compared to the pooled control and the solvent control. There was a statistical difference between the negative and solvent control for the second generation development time data; therefore, the treatment groups were compared to the negative control and solvent control separately. There were no statistically significant differences between the pooled control or the solvent control and the treatment group first generation development time data using a Bonferroni t-test (pooled control) or a Dunnett’s test (solvent control). During the second generation, the mean development time in the negative control, solvent control, 53, 108, 205, 432 and 827 mg/kg treatment groups was 15.9, 15.0, 15.3, 16.1, 15.8, 16.3 and 16.4 days, respectively (see Table 14, attached). There were no statistical differences between the negative control or solvent control and any of the treatment groups for the second generation development time data. Therefore, the NOEC for development time was 827 mg/kg, the highest concentration tested. The LOEC was >827 mg/kg.

 

Since there were no significant interactions between sex and treatment for the first generation development rate, the sexes were pooled for analysis. There were no statistical differences between the negative and solvent control development rates for the first generation data; therefore the treatment groups were compared to the pooled control and additionally, to the solvent control data. During the first generation, mean development rates (defined as the portion of larval development which takes place per day) in the negative control, solvent control, pooled control, 53, 108, 205, 432 and 827 mg/kg treatment groups were 0.0671, 0.0695, 0.0683, 0.0667, 0.0685, 0.0663, 0.0636 and 0.0634. There were statistically significant differences (p < 0.05) in mean development rate for the 432 and 827 mg/kg treatment groups during the first generation when compared to the pooled control using a Bonferroni t-test. There were also statistically significant differences (p < 0.05) in mean development rate for the 205, 432 and 827 mg/kg treatment groups during the first generation when compared to the solvent control using a Dunnett’s test. There was a statistical interaction between the treatments and sex for the second generation development rate data; therefore the development rate data for each sex was analyzed separately. The mean development rates in each control and treatment group are summarized in Tables 13 and 14 (attached). The mean male development rates for the second generation organisms were 0.0691, 0.0742, 0.0735, 0.0706, 0.0683, 0.0677 and 0.0678, respectively for the negative control, solvent control, 53, 108, 205, 432 and 827 mg/kg treatment groups. The mean female development rates for the second generation organisms were 0.0621, 0.0647, 0.0636, 0.0600, 0.0630, 0.0610 and 0.0607, respectively for the negative control, solvent control, 53, 108, 205, 432 and 827 mg/kg treatment groups.

 

There was a significant difference between the negative control and solvent control data for both sexes; therefore, the data for each sex was compared to the negative control and solvent control data separately. There were no significant differences in comparison of the second generation development rates between the negative control and the treatment groups using Dunnett’s test (p > 0.05) for either sex. There were statistically significant differences in the second generation male development rate in the 108, 205, 432 and 827 mg/kg treatment groups when compared to the solvent control using a Dunnett’s test. There were also statistically significant differences in the second generation female development rates in the 108, 432 and 827 mg/kg treatment groups when compared to the solvent control using a Dunnett’s test. Therefore, the NOEC for development rate was 53 mg/kg and the LOEC was 108 mg/kg, based on the significance observed in the second generation data.

 

The sex ratio was determined for each experimental group for each generation. In the first generation, the male sex ratio for the negative control, solvent control, 53, 108, 205, 432 and 827 mg/kg treatment groups was 0.40, 0.46, 0.37, 0.41, 0.46, 0.44 and 0.47, respectively. The female sex ratio was 0.57, 0.46, 0.57, 0.49, 0.47, 0.49 and 0.41, respectively. In the second generation, the male sex ratio for the negative control, solvent control, 53, 108, 205, 432 and 827 mg/kg treatment groups was 0.44, 0.48, 0.44, 0.45, 0.49, 0.47 and 0.38, respectively. The female sex ratio was 0.48, 0.44, 0.51, 0.54, 0.41, 0.49 and 0.44, respectively. The sex data was analyzed as the number of males that emerged in a treatment group compared to the total number of emerged adults in the group, using a Bonferroni t-test. No significant differences were observed between the pooled control and the treatment groups. Therefore, the NOEC and LOEC for sex ratio are 827 and >827 mg/kg, respectively.

 

The fecundity and fertility of the egg ropes produced by the first generation midges was also analyzed. There was a statistical difference in fecundity in the 205 mg/kg treatment group using a Jonckheere Terpstra trend test; however, the difference was not dose responsive and was therefore not considered to be treatment related. There were statistical differences in fertility in the 108, 205, 432 and 827 mg/kg treatment groups using a Jonckheer-Terpstra trend test. Therefore, the NOEC and LOEC for fertility of the egg ropes (most sensitive of the two endpoints) are 53 and 108 mg/kg, respectively. The NOEC and LOEC for fecundity of the egg ropes was 827 and >827 mg/kg, respectively.

 

CONCLUSION

Two generations of the midge (Chironomus riparius) were exposed for 28 days to five mean measured concentrations of sediment-incorporated test item ranging from 53 to 827 mg/kg. There were treatment-related effects observed on fertility of the egg ropes in the 108, 205, 432 and 827 mg/kg treatment groups. Fertility and development rate were the most sensitive endpoints. Based on the mean measured concentrations in sediment, the NOEC was 53 mg/kg and the LOEC was 108 mg/kg. The NOEC and LOEC for emergence ratio, development time, sex ratios and fecundity were 827 and >827 mg/kg, respectively.

Description of key information

Two generations of the midge (Chironomus riparius) were exposed for 28 days to five mean measured concentrations of sediment-incorporated test item ranging from 53 to 827 mg/kg. There were treatment-related effects observed on fertility of the egg ropes in the 108, 205, 432 and 827 mg/kg treatment groups. Fertility and development rate were the most sensitive endpoints. Based on the mean measured concentrations in sediment, the NOEC was 53 mg/kg and the LOEC was 108 mg/kg. The NOEC and LOEC for emergence ratio, development time, sex ratios and fecundity were 827 and >827 mg/kg, respectively (OECD 233).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

53 mg/kg sediment dw

Additional information

GUIDELINE

The study was based on procedures in the OECD Guidelines for the Testing of Chemicals, Guideline 233: Sediment-Water Chironomid Life-Cycle Toxicity Test Using Spiked Water or Spiked Sedimentand ASTM Standard E 1706-05: Standard Test Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates.

 

METHODS

The objective of this study was to determine the effects of sediment-incorporated test item on two generations of the midge,Chironomus riparius, during a 28-day exposure period under static test conditions for each generation. The evaluated endpoints for both the first and second generations were the total number of adults emerged, development rate, and sex ratio of fully emerged and alive adults. The number of egg ropes per female and the fertility of the egg ropes was also evaluated for the first generation.

 

Midges were exposed to a geometric series of five test concentrations, a negative control (untreated formulated sediment), and a solvent control (acetone) for 28 days under static test conditions for each of the two generations. Exposure in each generation consisted of eight replicate test chambers maintained in each treatment and control group, with 20 midges in each test chamber, for a total of 160 midges per test concentration, per generation. Each test chamber contained sediment and overlying water. An additional three replicates were prepared in each treatment and control group for use in analytical sampling (for each generation). No midges were placed in the additional replicates sampled on Day 0, but those sampled on Days 14 and 28 had midges added at the same time as the“biological”replicates on Day 0. These additional replicates were not used to evaluate the biological response of the test organisms.

 

Test concentrations in the sediment were prepared on a mg/kg dry weight basis. Nominal test concentrations were selected in consultation with the Sponsor based on exploratory range-finding toxicity data, and were 63, 125, 250, 500 and 1000 mg test item/kg of sediment. Test concentrations were measured in samples of overlying water, pore water, and sediment collected on Day 0, Day 14, and on Day 28 of each generation. Additionally, samples of overlying water pooled from the crystallizing dishes in each experimental group as well as sediment and pore water from each crystallizing dish were collected at the end of the first generation exposure. The results of the study are based on mean measured test concentrations in the sediment measured on Day 0 for both generations.

 

The water/sediment systems in the test compartments were allowed to equilibrate for approximately 48 hours prior to introduction of the organisms. First-instar, 1 to 3-day old midge larvae were impartially assigned to exposure chambers at test initiation. Observations of abnormal behavior were made daily throughout the test. In the first generation, chironomid emergence, time to emergence and sex ratio of the fully emerged and alive midges was assessed. Emerged adults were transferred to breeding cages (four replicates assigned to each of two cages, per experimental group) to facilitate swarming, mating and oviposition. The number of egg ropes produced and their fertility was assessed. From these egg ropes, first instar larvae were placed into freshly prepared test chambers to determine the viability of the second generation through an assessment of their emergence, time to emergence and the sex ratio of fully emerged and alive midges. Observations of the effects of the test item on the total number of adults emerged, development rate, and sex ratio of fully emerged and alive adults in both the first and second generations, as well as the number of egg ropes per female and fertility of the egg ropes from the first generation were used to determine the no-observed effect concentration (NOEC) and the lowest-observed effect concentration (LOEC).

 

RESULTS

No unusual observations of organisms avoiding the sediment occurred during the test. There were a few observations of larvae on the surface of the sediment or swimming in the water column during the study, but these did not appear to be dose-responsive, were comparable between the control and treatment groups, and were not considered to be treatment-related. There also were a few observations of dead pupae or larvae and adults that emerged and died. However, these numbers were small, were not concentration responsive, and were not considered to be treatment-related.

 

There were no apparent treatment-related effects on emergence at any concentration tested. During the first generation emergence was first noted on Day 12 of the test and continued through Day 24. The mean emergence ratios in the negative control, solvent control and the 53, 108, 205, 432 and 827 mg/kg treatment groups were 0.97, 0.92, 0.94, 0.91, 0.93, 0.94 and 0.88, respectively. During the second generation, emergence was first noted on Day 13 of the exposure and continued through Day 28. The mean emergence ratios in the negative control, solvent control, pooled control and the 53, 108, 205, 432 and 827 mg/kg treatment groups were 0.91, 0.91, 0.91, 0.96, 0.99, 0.90, 0.96 and 0.82, respectively. There were no significant differences in comparisons of emergence ratios between the negative control and the treatment groups or the solvent control and treatment groups using a Dunnett’s test (p > 0.05) for the first generation. There was a significant difference in comparisons of emergence ratios between the 0.827 mg/kg treatment group and the 108 and 432 mg/kg treatment groups using a Kruskal-Wallis test (p≤0.05) for the second generation; however, the difference was not considered to be biologically meaningful as there was no difference observed from the pooled control or the solvent control. Therefore, the NOEC for emergence ratio was 827 mg/kg, the highest concentration tested. The LOEC for emergence ratio was determined to be >827 mg/kg.

 

During the first generation, the mean development time in the negative control, solvent control, pooled control, 53, 108, 205, 432 and 827 mg/kg treatment groups was 15.6, 15.1, 15.3, 15.7, 15.2, 15.8, 16.4 and 16.5 days, respectively. Since there were no statistical difference between the negative and solvent control for the first generation development time data, the treatment groups were compared to the pooled control and the solvent control. There was a statistical difference between the negative and solvent control for the second generation development time data; therefore, the treatment groups were compared to the negative control and solvent control separately. There were no statistically significant differences between the pooled control or the solvent control and the treatment group first generation development time data using a Bonferroni t-test (pooled control) or a Dunnett’s test (solvent control). During the second generation, the mean development time in the negative control, solvent control, 53, 108, 205, 432 and 827 mg/kg treatment groups was 15.9, 15.0, 15.3, 16.1, 15.8, 16.3 and 16.4 days, respectively (see Table 14, attached). There were no statistical differences between the negative control or solvent control and any of the treatment groups for the second generation development time data. Therefore, the NOEC for development time was 827 mg/kg, the highest concentration tested. The LOEC was >827 mg/kg.

 

Since there were no significant interactions between sex and treatment for the first generation development rate, the sexes were pooled for analysis. There were no statistical differences between the negative and solvent control development rates for the first generation data; therefore the treatment groups were compared to the pooled control and additionally, to the solvent control data. During the first generation, mean development rates (defined as the portion of larval development which takes place per day) in the negative control, solvent control, pooled control, 53, 108, 205, 432 and 827 mg/kg treatment groups were 0.0671, 0.0695, 0.0683, 0.0667, 0.0685, 0.0663, 0.0636 and 0.0634. There were statistically significant differences (p < 0.05) in mean development rate for the 432 and 827 mg/kg treatment groups during the first generation when compared to the pooled control using a Bonferroni t-test. There were also statistically significant differences (p < 0.05) in mean development rate for the 205, 432 and 827 mg/kg treatment groups during the first generation when compared to the solvent control using a Dunnett’s test. There was a statistical interaction between the treatments and sex for the second generation development rate data; therefore the development rate data for each sex was analyzed separately. The mean development rates in each control and treatment group are summarized in Tables 13 and 14 (attached). The mean male development rates for the second generation organisms were 0.0691, 0.0742, 0.0735, 0.0706, 0.0683, 0.0677 and 0.0678, respectively for the negative control, solvent control, 53, 108, 205, 432 and 827 mg/kg treatment groups. The mean female development rates for the second generation organisms were 0.0621, 0.0647, 0.0636, 0.0600, 0.0630, 0.0610 and 0.0607, respectively for the negative control, solvent control, 53, 108, 205, 432 and 827 mg/kg treatment groups.

 

There was a significant difference between the negative control and solvent control data for both sexes; therefore, the data for each sex was compared to the negative control and solvent control data separately. There were no significant differences in comparison of the second generation development rates between the negative control and the treatment groups using Dunnett’s test (p > 0.05) for either sex. There were statistically significant differences in the second generation male development rate in the 108, 205, 432 and 827 mg/kg treatment groups when compared to the solvent control using a Dunnett’s test. There were also statistically significant differences in the second generation female development rates in the 108, 432 and 827 mg/kg treatment groups when compared to the solvent control using a Dunnett’s test. Therefore, the NOEC for development rate was 53 mg/kg and the LOEC was 108 mg/kg, based on the significance observed in the second generation data.

 

The sex ratio was determined for each experimental group for each generation. In the first generation, the male sex ratio for the negative control, solvent control, 53, 108, 205, 432 and 827 mg/kg treatment groups was 0.40, 0.46, 0.37, 0.41, 0.46, 0.44 and 0.47, respectively. The female sex ratio was 0.57, 0.46, 0.57, 0.49, 0.47, 0.49 and 0.41, respectively. In the second generation, the male sex ratio for the negative control, solvent control, 53, 108, 205, 432 and 827 mg/kg treatment groups was 0.44, 0.48, 0.44, 0.45, 0.49, 0.47 and 0.38, respectively. The female sex ratio was 0.48, 0.44, 0.51, 0.54, 0.41, 0.49 and 0.44, respectively. The sex data was analyzed as the number of males that emerged in a treatment group compared to the total number of emerged adults in the group, using a Bonferroni t-test. No significant differences were observed between the pooled control and the treatment groups. Therefore, the NOEC and LOEC for sex ratio are 827 and >827 mg/kg, respectively.

 

The fecundity and fertility of the egg ropes produced by the first generation midges was also analyzed. There was a statistical difference in fecundity in the 205 mg/kg treatment group using a Jonckheere Terpstra trend test; however, the difference was not dose responsive and was therefore not considered to be treatment related. There were statistical differences in fertility in the 108, 205, 432 and 827 mg/kg treatment groups using a Jonckheer-Terpstra trend test. Therefore, the NOEC and LOEC for fertility of the egg ropes (most sensitive of the two endpoints) are 53 and 108 mg/kg, respectively. The NOEC and LOEC for fecundity of the egg ropes was 827 and >827 mg/kg, respectively.

 

CONCLUSION

Two generations of the midge (Chironomus riparius) were exposed for 28 days to five mean measured concentrations of sediment-incorporated test item ranging from 53 to 827 mg/kg. There were treatment-related effects observed on fertility of the egg ropes in the 108, 205, 432 and 827 mg/kg treatment groups. Fertility and development rate were the most sensitive endpoints. Based on the mean measured concentrations in sediment, the NOEC was 53 mg/kg and the LOEC was 108 mg/kg. The NOEC and LOEC for emergence ratio, development time, sex ratios and fecundity were 827 and >827 mg/kg, respectively.