N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Apr 2003 to Nov 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: HARAP

Version / remarks:

Guidance document on higher-tier aquatic risk assessment for pesticides (HARAP), 1999

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Application solutions and storage stability samples were analysed for the test substance using the internal analytical method RAM 403/01.
Analytical intervals: Analytical samples were taken at the following intervals from all microcosms:1, 3, 6, 12 and 24 hours, 3, 7 and 14 days after the first application. 1, 3, 6, 12 and 24 hours, 3, 7, 14, 21 and 28 days after the second application. In addition, samples were taken from microcosms M03 and M11 (top rate treatments) at 35, 42, 49, 56 and 63 days after the second application.
Sampling method: On each sampling sampling occasion, 10 mL samples were removed from each of the shallow, medium and deep sections using a disposable pipette, taking care not to disturb the sediment. The three 10 mL samples were combined into a pre-labelled 250 mL polypropylene screw top tube containing 30 mL of acetonitrile from which a 20 mL single sample was analysed. All control microcosms that did not receive any test item application were only sampled once at 1h after the first application and one control microcosm (M01) was sampled up to and including 28 days after second application.

Vehicle:

no

Details on test solutions:

- Preparation: Two applications of the test substance were made to the microcosms with a 14-day interval. A stock solution of the test substance was prepared by taking 8 mL of the formulation (51.9 g test substance/L) and making it up to 800 mL with deionised water. Known volumes of this solution were then diluted with deionised water to give concentrations of the test substance appropriate for treating the microcosms at each of the treatment rates. For the first application, the test solutions were prepared the evening before application and stored overnight at 4°C in darkness (stability under these conditions had been previously verified). For the second application, the test solutions were prepared on the morning of application. Prior to addition to the microcosms, subsamples of the application solutions were taken to verify concentrations using the analytical procedures. Volume of application solution added to each section of the microcosms was as following:
Shallow microcosm: 140 mL
Medium microcosm: 530 mL
Deep microcosm: 830 mL
Total: 1500 mL
- Application: Application of the test substance was by direct addition in ascending concentration order i.e. 0.1 μg/l first and 100 μg/l last. Application of the test substance was carried out by 3 experimentalists (1 for each section of the microcosm), who poured the test substance from a measuring cylinder into the microcosm in a zigzag pattern over the water surface. Simultaneously, 3 other experimentalists (1 for each section of the microcosm) mixed the test substance in the water, using a rod, for one minute, taking care to minimise disturbance to the sediment. The measuring cylinders were rinsed with deionised water and the rinsings applied in the same manner. Deionised water was applied to the 4 control microcosms, and a 1-minute stirring period was made to each.

Test organisms (species):

other: algae communities, zooplankton communities, Macroinvertebrates and Macrophytes

Details on test organisms:

- Algae: Phytoplankton, periphyton and epiphyton populations were allowed to develop naturally within the microcosms and would also have been introduced with the macrophyte and macroinvertebrate additions.
- Zooplankton and Macroinvertebrates: In addition to those entering the microcosms with the water, sediment and plants, populations of zooplankton and macroinvertebrates were enhanced artificially by addition from the test facility’s Pond Site stock ponds and stock tanks. Two additions were made, on the 20 and 25 March 2003. Sweep nets (1 mm mesh) and zooplankton nets (100 μm mesh), were used to collect macroinvertebrates and zooplankton from the stock ponds and stock tanks from around the pond site. Netting took place in the water from amongst a variety of different plant stands, various water depths, and on the bottom sediment as well as from within the water column, and at the edges of the stock ponds and tanks as well as in the middle. The samples collected were placed into a clean mixing tank containing tap water. The contents of the tank were mixed continuously and distributed evenly between 24 buckets. The buckets were selected at random for positioning next to each of the 24 microcosms. Following this, the invertebrate-water mix was poured into the appropriate sections of the 24 microcosms.
- Macrophytes: A range of macrophytes was added to the microcosms with the species selection being consistent with the depth profile of the section, i.e. emergent species were planted in the shallow section, while floating and submerged plants were placed in the deeper sections. Planting was carried out uniformly in all 24 microcosms. Planting began on 18 March 2003 and was completed by 26 March 2003. Plants were obtained from both commercial and established pond sources. Those removed from established ponds at the test facility’s Pond Site were distributed evenly throughout the 24 microcosms. Plants brought in commercially from British Flora were extracted from their pots and planted evenly throughout the microcosms. Grass seed from Herbiseed, was sprinkled by hand onto the upper half of the bank in the shallow section of each microcosm. The plants were left undisturbed to establish naturally. The majority of macrophytes were planted in the shallow sections of the microcosms in order to provide a natural habitat to enhance growth and diversity of macroinvertebrates. During the course of the study other aquatic macrophytes emerged naturally in the microcosms, these were recorded and identified.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

129 d

Hardness:

- Control: 105.80 - 197.40 mg/L as CaCO3
- Treatment groups: 98.35 - 193.07 mg/L as CaCO3

Test temperature:

- Control: 9.8 - 22.1 °C (day - day 90)
- Treatment groups: 9.6 - 9.9 °C and 14.6 -14.8 °C (applications 1 (day 0) and 2 (day +14), respectively)
The difference in mean temperature between the control and other treatments was a maximum of 3.9°C on day +62 and less than 1.2°C on all other dates.

pH:

- Control: 7.03 - 9.56
- Treatment group: 7.21 - 10.04

Dissolved oxygen:

- Control: 53% - 131% of saturation
- Treatment groups: 72.34% - 137.75 % of saturation

Conductivity:

- Control: 292.50 - 529.75 μS/cm
- Treament groups: 289.19 - 524.67 μS/cm

Nominal and measured concentrations:

Nominal concentration: 0 (control), 0.1, 0.3, 1.0, 3.0, 100 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Outdoor microcosm. Each individual microcosm consisted of a rectangular fiberglass tank (1.0 m width, 4.5 m long) divided into 3 discrete sections of different depth and length. The depth and length of the 'shallow,' 'medium,' and 'deep,' sections of the microcosms were approximately 0.3 m x 2 m, 0.5 m x 1.5 m and 0.9 m x 1 m respectively. The microcosms were sunk into the ground with the top edge at ground level, in order to buffer and stabilise the temperature in the microcosms. A total of 24 microcosms were available, of which 20 were selected for use in the study.
- Type: Open
- No. of organisms per microcosm, No. of microcosm per concentration: See Table 2 in 'Any other information on materials and methods inlc. tables'
- No. of microcosm per control: 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water and sediment: Sediment was added to a depth of approximately 10 cm in each of the shallow, medium and deep sections, The microcosm construction incorporated a 10 cm barrier 'lip' on each of the shallow and medium section borders, to prevent the sediment falling in to the adjoining deeper section of the microcosm. Water was added to give depths of approximately 10 cm (shallow section), 30 cm (medium section) and 70 cm (deep section). The length of these sections was 2 m, 1.5 m and 1 m respectively. A mud bank was also incorporated into the shallow section, sloping from the top edge of the microcosm and extending approximately halfway along the section length. The mud bank was included to encourage certain aquatic organisms known to favour this environment e.g. Coleoptera using the bank to crawl out of the water to pupate in the mud.
The approximate water volume for the shallow, medium and deep sections were 117 L, 450 L and 700 L respectively, giving an overall microcosm capacity of approximately 1267 L. Mixing and addition of the sediment and water was carried out between the 11th and 18th March 2003. Sediment was transferred by wheelbarrow from source ponds 5 and 6 and distributed between the microcosms to give a depth of approximately 10 cm in each section. The sediment was levelled with a rake and water from source pond 4 was added to cover and protect the sediment from frost damage.


OTHER CONDITIONS
- Water level: The water levels in the microcosms were monitored weekly throughout the study period. A piece of tape was placed on the microcosm lip, 1 m down from the bank end of the shallow section of each microcosm to mark the positioning for water depth measurements. Water depth, to the nearest centimeter, was measured at this position in the microcosms using a ruler placed in front of the tape marker.
Water level adjustments were made when fluctuations were +/- 5 cm from the desired level, or when considered necessary. Microcosm water additions were made to the deep section using water pumped from source pond, filtered through a 100 μm mesh filter. Where water needed to be removed, the water was be extracted and poured down the drain. The only exceptions to this were on study days +24 (6th June 2003) and +64 (16th July 2003) where water from source pond was not filtered and added to the following microcosms; day +24: M09, M10, M14, M18, M19, M20, M21 and M24 and day +64: M06, M08 to M12, M14 to M21, M23 and M24. Plankton data were compared for replicates that received unfiltered and filtered water and it was concluded that the addition of unfiltered water had no impact on the data.
- Macrophytes: The microcosms were set up to include a natural shallow zone providing a good cover of marginal vegetation, in addition to the deeper areas supporting lower numbers of macrophytes. The different sections aimed to provide a range of habitats and enhance the diversity of flora and fauna in the systems. Microcosms were prepared as similarly as possible. A list of species present in the microcosms was prepared on the 17th April and 19th September 2003. The percentage cover (when viewed from above) of submerged/floating/emergent plants, open water and bare-ground was also visually assessed on these days. Assessments were made in 5% increments with any group present at <5% being scored as <5%.
- Meteorological Conditions: Rainfall and air temperature readings were recorded weekly during the study. Rainfall was determined by a manual rain gauge measuring rain in graduations of 1 mm/m2. A push button max-min thermometer was used to record the air temperature. For the first application (13/5/03) it was sunny, cloudy and breezy. During the second application (27/5/03) it was cloudy with a light wind.

EFFECT PARAMETERS MEASURED: Sampling schedule is provided in Table 3 in 'Any other information on materials and methods incl. tables'. On each sampling occasion, the deep section of each microcosm was sampled using a mechanical depth integrating, trap door water column sampler. The sampler was lowered vertically through the water column, with the entrance flap open, until it was approximately 10 cm above the sediment. The sampler was then sealed, by pulling the lever-handle, and the entire water column then lifted vertically from the microcosm. If a large amount of filamentous algae was present, the sample was discarded and another sample taken. The sampler contents were then poured into a bucket. This was repeated until approximately 12 liters were collected in the bucket. The combined water sample was mixed vigorously with a wooden stirrer and sub-samples were taken for analysis. Excess water was then returned carefully to the deep section of the sampled microcosm.
- Chlorophyll-a analysis: A 500 mL, appropriately labelled bottle, was immersed in the combined water column sample, and the bottle was filled and then capped. The sample was taken to the laboratory to be processed as soon as possible for alkalinity, hardness and chlorophyll-a determinations. Chlorophyll-a was determined by fluorometry, which was initially calibrated using
HPLC.
- Phytoplankton: A 250 mL, appropriately labelled bottle, was immersed in the combined water column sample, and the bottle was filled and then capped. The sample was taken to the laboratory to be processed and preserved as soon as possible by adding 0.5 to 1 mL of Lugol's-iodine solution to the sample bottle, to preserve the phytoplankton. Samples were transferred for analysis.
- Zooplankton: A 12 L subsample of the microcosm water was extracted from the composite water column sample using a plastic measuring jug, and poured through a 100 μm mesh size conical plankton net with detachable end sieve. The inside of the plankton net was washed into the end sieve with tap water from a low-pressure hose. The end sieve was then detached from the net and the contents washed into a 125 mL sample bottle with tap water from a wasbottle. The amount of water used was kept to a minimum, so that the bottle was a quarter to one third full. The sample was taken to the laboratory to be processed and preserved as soon as possible by topping up the volume with IMS to give approximately a 70% aqueous IMS solution. Two drops of 0.4% Rose Bengal stain were then added. Samples were prepared for identification and enumeration according to MESOCOSM GmbH SOP/015/04 with the following exception. SOP/015/04 states formaldehyde as the reagent for preserving zooplankton samples. In this study the samples were preserved. The zooplankton sample was filtered through a gauze of 63 μm mesh size. Organisms remaining on the gauze were rinsed off with water into a counting chamber along with any organisms remaining in the sample bottle or lid. The samples were evaluated under a stereomicroscope with transmitted light illumination. The following groups were identified and counted in the sample: Rotatoria, Cladocera, Copepoda and Ostracoda. All adult Crustacea (Cladocera, Copepoda and Ostracoda) were identified to species level if this was possible without extensive preparation of the organisms. No size classifications or egg counts were made, with the exception of the Copepoda for which counts of nauplii were included.
Following identification and enumeration, the number of individuals per liter of each taxon was determined taking into consideration the sampling volume, the counted area (usually between 25 and 50% of the total counting chamber area) and the total area of the counting chamber.
- Macroinvertebrates: Macroinvertebrate sampling was carried out using 3 techniques:
1. Enhanced Surface Area Substrate samplers (ESAS) - artificial colonising
substrates to sample benthic and epiphytic dwellers.
2. Sweep-netting (NETS) - to sample swimming and epiphytic organisms.
3. Emergence Traps (ET) - to sample emergent adult insects.
Due to the small size of the test system and the resultant potential for destructive sampling of the populations, the organisms collected by ESAS and NETS were identified and enumerated live. The organisms were then returned to the originating microcosm.

Reference substance (positive control):

no

Key result

Duration:

129 d

Dose descriptor:

other: NOAEC

Effect conc.:

0.3 µg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Ecological effects of various community and population endpoints

Details on results:

An overview of the results is provided in Table 4 - Table 6 in 'Any other information on results incl. tables'.

- Chlorophyll-a: Chlorophyll-a concentrations in control microcosms ranged from 1.01 μg/L on day +48 to 4.52 μg/L on day +97. There was an isolated non-systematic significant (p ≤ 0.05) reduction in the 0.3 μg test substance/L treatment on day +20. A significantly increased chlorophyll-a concentration was observed at the highest treatment (100 μg test substance/L) on day +41. There was however a trend for a slightly higher concentration following treatment, although chlorophyll-a concentrations were also high in the 100 μg test substance/L treatment prior to application. The variation observed in all other treatments up to and including the 3 μg test substance/L treatment was apart from individual isolated dates within the range observed in the control microcosms.

- Overall results for phytoplankton: No significant direct and treatment related effects on phytoplankton communities based on a total of 186 taxa were observed in any of the treatments up to and including the 100 μg test substance/L treatment. Later in the study from about day +51 onwards, the Principal Response Curves (PRCs) indicated changes in the phytoplankton communities. However, these changes led only to isolated significant (p ≤ 0.05) reductions observed in both the 0.1 and 0.3 μg test substance/L treatments. Significant (p ≤ 0.05) reductions were observed over continuing time periods in the 3 μg test substance/L treatment from days +62 to + 76 and in the 100 μg test substance/L treatment from days +69 onwards for the rest of the study until day +125. The only taxon clearly reflecting the above-mentioned significant reductions in the PRCs at higher treatment rates during the later part of the study was Nitzschia palea. Nitzschia palea showed no significant (p ≤ 0.05) changes in abundance in the 0.1 μg test substance/L treatment and only isolated significant reductions in the 0.3 μg test substance/L treatment. Similar reductions as for Nitzschia palea were also seen in Lyngbia spec., Oscillatoria rosea and Oscillatoria spec., however all these taxa showed no or only isolated significant differences. The fact that these four taxa made up about 37% of the overall abundance (based on the control microcosms) may explain the observed changes in the PRCs towards the end of the study. An opposite picture with significantly increased abundances in the two highest treatments (3 and 100 μg test substance/L) during the later part of the study was observed in Volvox aureus cf. Six out of the 23 taxa analysed using univariate statistics showed significant density increases during a time period directly following the application. For Synura spec., Chromulina ovalaides cf., Oscillatoria spec., Euglena clavata cf. and Trachelomonas volvocina cf. were these changes only observed in the two highest (3 and 100 μg test substance/L) treatments, while Monoraphidium circinale also showed a single significant (p ≤ 0.05) increase in the 1 μg test substance/L treatment on day +6. Furthermore, return to control levels was completed in all species by day +62, i.e. within 48 days following the last application. A total of nine taxa showed only isolated or not dose-related significant changes in their abundances (Chlorella vulgaris, Characium, spec., Clamydomonas spec., Chromulina minima cf., Gomphonema angustatum cf., Cryptomonas erosa + ovata, Anabaena spec., Rhodomonas spec., and Oscillatoria rosea). Of these taxa, Rhodomonas spec. showed one period of three subsequent sampling dates (days +62 to +76) with a significantly (p ~ 0.05) reduced abundance in the 3 μg test substance/L treatment, which was, however, not present in the higher 100 μg test substance/L treatment. A further 6 out of the 23 taxa analysed using univariate statistics showed no significant (p ≤ 0.05) changes during any time following the application. These were the taxa Coelastrum microporum, Scenedesmus aculeolatus, Clamydomonas gloeophila cf., Achnanthes minutissima, Chrysophyceae (2-3 μm), and Lyngbia spec. Given the conservative nature of the test system and considering the fact that observed changes were either short-term or not directly related to application, it is concluded that no effects were observed at 0.1 μg test substance/L. No ecologically unacceptable effects on the phytoplankton community structure or on individual populations were seen after 2 applications up to and including 0.3 μg test substance/L.

- Overall results for zooplankton: Two applications of the test substance resulted in significantly (p ≤ 0.05) altered zooplankton community structure for all treatment rates used in this study. Communities in the two lowest treatments (0.1 and 0.3 μg test substance/L) recovered rapidly (27 days following application for the 0.1 μg test substance/L treatment and 20 days for the 0.3 μg test substance/L treatment) to a level, which was again closer to the control. For most of the remaining study period PRCs for both treatments indicated a temporal pattern of changes between statistical significant and not significant data points. It is assumed that this indicates a slowly but steadily continuing recovery process. This assumption is supported by the fact that community structures in both treatments were close to the control with no significant differences over the last three sampling dates of the study. The two applications of the test substance caused a more pronounced effect in the 1 and 3 μg test substance/L treatments. While there were still some signs of recovery, such as periods of no significant differences, observed in the 1 μg test substance/L treatment, the 3 μg test substance/L treatment had a significantly (p ≤ 0.05) different community structure for most of the post application period apart from a few exceptions. A generally higher variability was observed in the highest 100 μg test substance/L treatment. Although the absolute differences in the PRCs between this treatment and the control were highest, there were only a few dates with significant differences. Taxa belonging to the order Copepoda generally showed the highest sensitivity. However, even those taxa that had the highest significant (p ≤ 0.05) weights in the PRC (Copepodite and Nauplia) only showed transiently reduced population densities following application in all treatment rates up to and including the 100 μg test substance/L treatment. Recovery was completed in all treatments up to and including the 1 μg test substance/L treatment by day +48, i.e. within 34 days following the second application. Recovery in the 3 μg test substance/L treatment was completed for both taxa within 55 days following the second application. Diaptomid were not affected in the lowest treatment rate (0.1 μg test substance/L). Higher treatments showed no consistent pattern of significantly (p ~ 0.05) reduced densities and most of the significance differences observed in both the 0.3 and 1 μg test substance/L treatments were a result of isolated peaks in control abundances. The significantly altered densities observed for Cyclopidae in all treatment rates between days +20 and +41 can also be seen as a result of transiently elevated control densities. However, densities for Diaptomidae and Cyclopidae were generally very low leading to arbitrary significances, which cannot be related reliably to applications. The cladoceran Chydorus sphaericus showed a stronger population increase in the higher treatment rates, which is due to the absence of a clear dosedependence most likely an indirect reaction related to the dynamic of other plankton taxa rather than a result of the test substance application. Other cladocerans, such as daphnids were significantly (p ≤ 0.05) affected only in the two highest treatment rates (3 and 100 μg test substance/L) or showed a very rapid recovery within 13 days following application. The rotifers Keratella quadrata and Synchaeta spec. both showed only isolated non-systematic significant changes in their population density. It is concluded that the effects observed at the two lowest treatment rates 0.1 and 0.3 μg test substance/L were either transient with rapid recovery or a result of very low population densities leading to arbitrarily significant values. Various population effects were seen in the 1 μg test substance/L treatment, however they also showed recovery within 34 days following the second application in almost all of the cases. Clear dose-related and longer lasting effects were seen in the two highest treatments at both the community and the population level. Considering the overall picture derived from the zooplankton data, the test substance concentration at which no ecologically unacceptable effects were present following two applications is 0.3 μg test substance/L.

- Overall results of macroinvertebrates: Two applications of the test substance had no significant (p ≤ 0.05) effect on the macroinvertebrate community structure in the lowest 0.1 μg test substance/L treatment rate. Community structure was affected in the higher treatments but showed a rapid recovery within 36 days following the last application in the 0.3 μg test substance/L treatment and a slower recovery within 65 days following the last application in the 1 μg test substance/L treatment. Only very little or no recovery was seen in the higher treatment rates and significant differences were sustained to the end of the study. Crustaceans showed the strongest reactions to the two applications among all macroinvertebrates sampled. The abundance of the isopod Asellus aquaticus was significantly reduced as a direct result of the applications in the two highest treatment rates. Recovery was present in the 3 μg test substance/L treatment within 65 days following the last application. Only individual isolated and non-systematic effects were observed in the lower treatment rates up to and including the 1 μg test substance/L treatment. The amphipod Crangonyx pseudogracilis showed no effects in the lowest 0.1 μg test substance/L treatment. Abundances were reduced in the 0.3 μg test substance/L treatment, however recovery within 51 days was apparent in this treatment rate from the sweep net samples. A marked decrease in the abundances with no or only little recovery was seen in the three highest treatments (1, 3 and 100 μg test substance/L). The reduced recovery rates of both the amphipod and the isopod taxon were a result of the fact that their entire populations were exposed to the test substance in the microcosms due to the conservative toxicological exposure scenario. The resulting lack of refugia within the system caused very strong effects on both taxa with their completely aquatic life cycles.
Population level analysis indicated no or only single isolated significant (p ≤ 0.05) effects for the molluscan species (e.g. Physa spec., Planorbarius corneus, Lymnea spec. or Musculium lacustre) in all treatment rates up to and including the 3 μg test substance/L treatment. The same applies to the annelid species Lumbriculus variegatus and Tubifex spec., which both showed significantly increased densities only in the one or two highest treatments. Planariidae were not affected in any of the concentrations applied. Most of the insect taxa were either affected significantly (p ≤ 0.05) only in the highest 100 μg test substance/L treatment (e.g. Cloeon dipterum, Chironomidae, Gerris spec., Notonecta spec., Hydrophilidae adult, Diptera pupae) or in none of the treatments up to and including 100 μg test substance/L (Trichoptera). Coenagrionidae were significantly reduced in the 100 μg test substance/L treatment, however the nets samples indicated significantly elevated densities in all lower treatment rates during a period confined to a seasonal population growth from July onwards. Due to very low overall population densities, individual significant (p ≤ 0.05) reductions in abundances were seen for the hemipterans Corixidae in all treatment rates. Recovery was very rapid, i.e. within 23 days following the second application. The Chaoboridae showed no effects at 0.1 μg test substance/L and significantly reduced population densities with recovery 37 days after the second application in the 0.3, 1 and 3 μg test substance/L treatments. No direct treatment-related effects were visible on the emergence of semiaquatic insects in the two lowest treatment rates of 0.1 and 0.3 μg test substance/L. Isolated alterations of the community structure were observed in the 1 and 3 μg test substance/L treatments, however they were only present on single dates. Community emergence recovered in the two highest treatment rates within 42 days following the last application. Chironomidae as the most abundant group in the emergence samples showed no effect in the microcosms up to including the 0.3 μg test substance/L treatment and rapid recovery within 14 days following the last application in all other treatments. Given the conservative nature of the test system and considering the fact that observed changes were either short-term or not directly related to application, it is concluded that no effects were observed at 0.1 μg test substance/L. No ecologically unacceptable effects on the macroinvertebrate community structure or on individual populations were seen after 2 applications up to and including 0.3 μg test substance/L.

Reported statistics and error estimates:

All analyses were conducted using SAS, version 8.2. Following suitable transformation of the data to normalise the variance, multivariate and univariate techniques were applied to the data, as appropriate, to determine effects respectively on communities and populations of phytoplankton, zooplankton and macroinvertebrates. Univariate analysis (on transformed data where appropriate) was also applied to the physico-chemical data. Tests were carried out at the five percent probability level (p ≤ 0.05) and significant differences between treatments and the control are illustrated in all tables.

Table 4. Mean Chlorophyll a concentration (μg/L)

Day		Concentration (µg test substance/L)
	Control	0.1	0.3	1	3	100
Detransformed Adjusted Means (no./L)
-21	1.20	2.27	1.00	2.10	1.33	2.06
-15	1.77	1.79	1.35	2.43	1.88	2.86
-7	2.26	2.04	2.57	1.90	1.93	2.14
-1	1.93	1.88	1.00	1.29	1.00	1.98
6	2.67	1.72	2.27	2.26	2.43	2.33
10	1.88	1.75	1.68	2.01	2.77	3.85
20	2.00	1.46	1.00	1.75	2.21	1.77
27	3.97	2.44	1.44	1.88	3.37	4.40
34	1.90	1.62	1.70	1.13	2.79	4.96
41	1.42	2.25	1.70	1.39	3.21	10.40
48	1.01	1.00	1.00	1.00	1.00	1.33
55	2.80	1.30	2.10	1.80	1.42	5.50
62	2.06	2.36	1.75	1.93	1.56	4.38
69	2.56	4.82	1.91	2.57	1.67	4.06
76	3.62	2.06	2.73	1.83	2.22	3.94
83	2.94	1.29	1.64	1.13	2.85	4.59
90	2.86	1.22	1.90	1.55	1.90	3.85
97	4.52	1.86	3.50	5.20	4.07	8.94
105	1.17	1.00	1.04	1.82	1.00	2.26
111	4.37	2.49	3.13	2.76	3.56	4.64
118	3.63	2.91	2.70	3.91	3.73	11.61
125	3.82	2.03	4.34	3.08	3.32	15.58

Table 5. Mean abundance of Total Zooplankton

Day		Concentration (µg test substance/L)
	Control	0.1	0.3	1	3	100
Detransformed Adjusted Means (no./L)
-21	144.10	118.75	56.63	66.43	72.48	127.63
-15	70.70	112.43	58.4.8	135.84	98.46	111.55
-7	62.59	69.23	102.91	95.00	114.66	123.71
-1	147.55	156.69	243.85	362.96	296.25	213.36
6	62.87	115.55	85.81	59.42	170.08	98.25
10	99.95	103.83	128.93	42.61	371.03	112.08
20	108.05	113.01	122.10	299.39	158.40	30.14
27	127.57	186.69	196.68	162.49	143.99	13.40
34	64.40	103.39	98.47	124.41	188.66	4.58
41	243.93	191.83	189.55	259.78	573.65	87.12
48	73.64	72.23	126.81	107.53	109.80	221.12
55	49.03	62.70	54.74	74.97	183.84	497.46
62	68.43	140.01	54.41	105.98	83.11	1243.91
69	53.08	164.54	60.66	214.03	122.36	669.80
76	58.97	187.62	30.57	85.90	85.59	26.21
83	45.93	52.27	53.18	98.79	70.92	107.29
90	84.35	86.51	154.47	62.07	75.07	335.13
97	61.04	44.48	81.18	57.83	73.22	207.09
105	83.67	59.16	66.77	63.90	98.31	255.78
111	50.82	53.10	136.20	103.39	119.23	389.35
118	148.98	62.00	48.17	54.74	80.29	176.02
125	122.75	37.13	98.90	115.35	97.23	198.50

Table 6. Summary of population and community responses observed in the microcosm study

	Treatment levels ( µg test substance/L nominal)
	0.1	0.3	1	3	100
Population responses
Insecta (a)	2↓	2↓	2↓	2↓	2↓
Macro-Crustacea (b)	1	3↓	5↓	5↓	5↓
Other macroinvertebrates (c)	1	1	1	5↑	5↑
Micro-crustacea (d)	2↓	3↓	3↓	3↓	4↓
Other zooplankton (Rotifera) (e)	2↓	2↓	2↓	3↑	4↑↓
Phytoplanktonf	1	2↓	3↓	4↓	4↓
Macrophytes	1	1	1	1	1
Community responses
Community metabolism	1	1	1	1	3↓
Macro-invertebrates (PRC)	1	3↓	4↓	5↓	5↓
Zooplankton (PRC)	3↓	3↓	4↓	5↓	5↓
Phytoplankton (PRC)	1	2↓	3↓	4↓	5↓

Within each category the most sensitive endpoint was used.

↑ = increase, ↓ = decrease. Classification for population response based on: a Corixidae (nets), b Crangonyx pseudogracilis (ESAS), c

Tubifex spec. (ESAS), d Copepodites, e Keratella quadrata, f Nitzschia palea.

Class 1: “effect could not be demonstrated”

• no (statistically significant) effects observed as result of the treatment, and

• observed differences between treatment and controls show no clear causal relationship.

Class 2: “slight effect”

• effects reported in terms of “slight” or “transient” and/or other similar descriptions, and

• short-term and/or quantitatively restricted response of sensitive endpoints, and

• effects only observed at individual samplings.

Class 3: “pronounced short-term effect”

• clear response of sensitive endpoints, but total recovery within 8 weeks after the last application, and

• effects reported as “temporary effects on several sensitive species”, “temporary” elimination of sensitive species”, “temporary effects on less sensitive species/endpoints” and/or other similar descriptions, and

• effects observed at some subsequent sampling instances.

Class 4: “pronounced effect in short-term study”

• clear effects (such as strong reductions in densities of sensitive species) observed, but the study is too short to demonstrate complete recovery within 8 weeks after the (last) application.

Class 5: “pronounced long-term effect”

• clear response of sensitive endpoints and recovery time of sensitive endpoints is longer than 8 weeks after the last application, and

• effects reported as “long-term effects on many sensitive species/endpoints”, “elimination of sensitive species”, “effects on less sensitive species/endpoints” and/or other similar descriptions, and

• effects observed at various subsequent samplings.

Validity criteria fulfilled:

yes

Conclusions:

In an outdoor microcosm study, performed in accordanc with guideline document of Higher Tier Aquatic Risk Assessment for Pesticides (HARAP), the NOAEC for ecological effects of various community and population endpoints was 0.3 µg/L.

Executive summary:

The toxicity of the test substance on aquatic invertebrates was investigated in an outdoor microcosm study. The study was conducted according to the guideline document HARAP(Higher Tier Aquatic Risk Assessment for Pesticides) (1999) and was in compliance with GLP criteria. The test systems were rectangular varied depth microcosms, each containing approximately 1.3 m3 of water over 10 cm of sediment, with established communities of plants and invertebrates. The replicated study design consisted of five treatments (0.1, 0.3, 1.0, 3.0 and 100 μg test substance/L) and an untreated control. The treatment levels were chosen to incorporate known toxicity levels from laboratory studies and the maximum predicted environmental concentrations from proposed use patterns, and the 100µg test substance /L (highest) rate was included as it was considered most likely to give clear effects on a variety of taxa. Two applications of each treatment were made, two weeks apart, during May 2003 (13th and 27th). Application was by direct addition to the microcosms followed by thorough mixing to rapidly distribute the formulation through the water Column.

Physico-chemical properties of the water and phytoplankton, zooplankton and macroinvertebrate communities were studied for approximately one month prior to application and for approximately four months after application (126 days after 1st application). Water samples were collected for zooplankton, phytoplankton, chlorophyll a, alkalinity and hardness measurements. The multivariate Principal Response Curves method was employed to identify treatment-related changes in community structure. Analysis of variance was also used to look at population-level differences for selected taxa; those not analysed were considered to be too variable in occurrence or not abundant enough to permit meaningful conclusions to be drawn.

Chemical analysis for the test substance confirmed the nominal concentrations. The application solutions ranged between 90-106% of nominal (mean 100%). Thus, appropriate dosing was considered to have been achieved. Two applications of the test substance at 0.1 or 0.3 μg test substance/L had no direct significant treatment-related effects (p ≤ 0.05) on any of the physico-chemical parameters, on phytoplankton and macroinvertebrate community structure (PRC) or population dynamics (univariate statistics) measured in the study. Alterations of phytoplankton community structure or population densities of phytoplankton were, however, observed later during the study and were thus not directly related to application. Single and isolated effects were seen in a few macroinvertebrate taxa, and these were not considered to be treatment related. Zooplankton community structure and sensitive copepod taxa recovered within 34 days following the last application. All these effects are thus not considered significant adverse effects. Macroinvertebrate community structure and the abundance of the amphipod species Crangonyx pseudogracilis were affected in the 0.3 μg test substance/L treatment, but recovered within 36 days and 51 days following the last application, respectively. Two applications of the test substance at 1 μg test substance/L resulted in more pronounced and longer lasting effects on Nitzschia palea, zooplankton community structure and Crangonyx pseudogracilis from ESAS and nets samples. These effects were present for the majority of the post-application study period and they were sustained until the end of the study for Crangonyx pseudogracilis. In the 3 μg test substance/L treatment, the macroinvertebrate community structures based on both ESAS and nets samples indicated significant (p ≤ 0.05) treatment level effects over almost the whole post-application period, while the other pronounced and longer lasting effects observed at 1 μg test substance/L treatment were also present at 3 μg test substance/L. In addition to the already described effects, a number of even more pronounced and longer lasting effects were observed in the highest ( 100 μg test substance/L) treatment rate, namely increased densities of the annelid taxa Lumbriculus variegatus and Tubifex sp. in ESAS and nets samples, reduced densities of Asellus aquaticus, Coenagrionidae and Cloeon dipterum.

Considering the conservative nature of the study design and the type of effects observed, it was concluded that the two lowest treatments 0.1 and 0.3 µg test substance/L will not cause unacceptable ecological effects. Thus, the NOAEC value of the test substance is 0.3 μg test substance/L. Clear, dose-related and longer lasting effects were seen for a considerable number of endpoints in the two highest treatments 3 and 100 μg test substance/L. The 1 μg test substance/L treatment showed an intermediate response, with various community and population endpoints already affected.

Description of key information

All available data was assessed. The study representing the worst-case effects was included here and its effect value was used as the key value. Another study is included as supporting information.

Outdoor freshwater microcosm, 129-d NOAEC = 0.0003 mg/L, aquatic invertebrate communities, ecological effects, HARAP guideline, Schulz 2004

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Remarks:

NOAEC

Effect concentration:

0.3 µg/L

Additional information

There are two standard guideline followed and GLP complied studies available for this endpoint. The outdoor microcosm study (Schulz 2004; HARAP guideline; Reliability 1) was selected as key study, because it represents the worst-case effects (i.e. showed lower NOEC value) and closer to real-life condition than laboratory study. In the microcosm study, a formulation containing the test susbatcne was used. The microcosms were rectangular varied depth microcosms, each containing approximately 1.3 m3 of water over 10 cm of sediment, with established communities of plants and invertebrates. The replicated study design consisted of five treatments (0.1, 0.3, 1.0, 3.0 and 100 μg as/L) and an untreated control. Two applications of each treatment were made, two weeks apart, during May 2003 (13th and 27th). Phytoplankton, zooplankton and macroinvertebrate communities were studied for approximately one month prior to application and for approximately four months after application (126 days after 1st application). Water samples were collected for zooplankton, phytoplankton, chlorophyll, alkalinity and hardness measurements. The results show that the two lowest treatments 0.1 and 0.3 µg as/L will not cause unacceptable ecological effects. Thus, the NOAEC value of the test substance is 0.3 μg as/L.

The supporting study (Rufli 1987; OECD TG 202; Reliability 2) is a toxicity study on the reproduction of Daphnia magna using a semi-static system for 21 days. The test organisms (first instar < 24-hours old) were exposed to the test substance in reconstituted test water at 20 ± 1 ˚C and 16 hours light : 8 hours dark photoperiod (approx. 300 Lux). Five concentrations of nominally 0.0000010, 0.0000032, 0.000010, 0.000032, and 0.00010 mg/L were tested. The control group of animals was exposed to dilution water only and an additional vehicle control to the solvent at the concentration used for the highest test concentration (0.0095 mg DMF/L plus 0.00004 mg Alkylphenolpolyglycolether/L). Analytical investigations showed that all treatment lebels were below the detechtion limit of 0.0001 mg/L at days 0, 7, 14 and 21. The parental Daphnids, mortality occurred in all test substance treatment levels > 20% threshold. Accordingly, the NOEC values of for the Daphnia reproduction is not determinable according to the test guideline.