mecoprop-P (ISO); (R)-2-(4-chloro-2-methylphenoxy)propionic acid

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 November 2014 to 29 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

During the test period, the application solutions used for the dosage of the test media were used for a maximum of eight days before being renewed. The stability of the test material in the solvent was investigated in a pre-experiment and was confirmed within the definitive test.
From the freshly prepared application solutions, duplicate samples were taken on three preparation dates (Days -2, 26, and 48). To confirm the stability of the test material in the application solutions during their renewal periods, duplicate samples were taken from the aged application solutions on Days 5, 33 and 56, corresponding to renewal periods of seven and eight days.
From the test media, duplicate 10 mL samples were taken from all test concentrations and the solvent control at 26 dates starting from Day 0 until test end. At the different sampling dates, sampling was cycled from one replicate to the next. Thereafter, all samples were stored deep frozen (at about -20 °C). In pre-experiments for investigation of the storage stability of the samples (non-GLP), the test material proved to be stable under these storage conditions.
Only the highest test concentration of nominal 12 mg/L was analytically verified, as this test concentration was determined to be the NOEC in this test. From this test concentration, one of the duplicate samples from all sampling times was analysed.
From the corresponding application solution one of the duplicate samples was analysed from two stability controls (freshly prepared and aged application solutions). Thereby one of the stability controls corresponded to an eight days renewal period. From the control, one of the duplicate samples was analysed from 12 sampling dates during the test period.
The samples from all lower test concentrations were generally not analysed, since these concentrations were below the overall NOEC determined in this test, and thus, not relevant for the interpretation of the biological results. However, for verification of the dosing system, one sample from all other concentrations (nominal 0.12 to 3.8 mg/L) was analysed on Days 0, 5, and 7.

Vehicle:

yes

Remarks:

3,4 dimethylformamide

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The preparation of the test media was based on the OECD series on testing and assessment No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

In this flow-through test, different concentrations of the test material in the test media were maintained by dosing application solutions (concentrated solutions of the test material in 3,4 dimethylformamide analytical grade (DMF) into the test water using an automatic dosing system.
The application solutions were regularly renewed during the test period. The stability of the test material in water during the application solution renewal period of a maximum 8 days was determined in a pre-experiment and was confirmed by analytical measurements within this study.

- Method
The application solution of 195 g/L, used for the dosage of the highest test concentration of nominal 12 mg/L, was freshly prepared prior to each application solution renewal by completely dissolving the test material in DMF via 20 minutes of intensive stirring. Depending on the demand of application solution, nominal 78.0, 58.8 and 48.75 g of the test material were dissolved in 400, 300, and 250 mL DMF, respectively (the deviation of actual weighed test material was < 0.12 % of nominal). Adequate volumes of this application solution were diluted with DMF to obtain the application solutions used for the dosage of the lower test concentrations.
The application solutions were protected from light and were continuously stirred by magnetic stirrers. They were dosed automatically by a timer-controlled HAMILTON digital dispenser unit into a mixing vessel with a volume of about 2 L (dosage: 800 μL application solution per hour; 20 dosages per hour; 40 μL application solution per dosage). Test water flowed at a constant rate of 13 L/hour (312 L/day, controlled by a flow-meter) into the mixing vessels, where the test water and application solutions were continuously mixed together using intensive stirring with a magnetic stirrer.
From the mixing vessel, the test media flowed into electronically regulated splitting devices and were divided into four identical volumes. These volumes were directed into the corresponding four replicate test vessels of each treatment by teflon tubes.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: The study was started with newly fertilised eggs. After fertilisation of the eggs by the supplier, a batch of about 1000 eggs was immediately transferred to the testing facility using a cooled container.
- Subsequent handling of eggs: Before the test start, the eggs were acclimated to the test water for about 30 minutes. Then, the eggs were introduced into the incubation cups (= start of exposition to the test media). The time period between fertilisation and the introduction into the incubation cups was about three to four hours, considered to be sufficiently short.

POST-HATCH FEEDING
- Start date: During embryo-stage and for newly hatched larvae no food was supplied. After the yolk sac had been consumed by some of the fish, food was provided to the fish.
- Type/source of feed: The fish were fed with a commercial trout fish food.
- Amount given: Food was provided at least twice each working day. Food was given to the juvenile fish ad libitum while minimising the surplus. On weekends, food was given twice per day. Fish were not fed for 24 hours prior to the end of the test to allow clearance of the digestive tract before weighing the fish.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

89 d

Post exposure observation period:

Test duration was in total 89 days (hatching period from Day 0 to Day 29, plus a 60-day post hatch period, whereby Day 29 post fertilisation equals Day 0 post hatch).

Hardness:

214 - 232 mg/L as CaCO3.

Test temperature:

The mean water temperature in the different treatments during the test was in the range of 11.6 to 12.3 °C. The mean water temperature in the controls and at the different test concentrations over the course of the test was 11.9 °C

pH:

The pH values in the controls and at the test concentrations ranged between 7.8 and 8.1.

Dissolved oxygen:

During the test period, the dissolved oxygen concentration in the controls and at the test concentrations was at least 7.8 mg/L, corresponding to an oxygen saturation of at least 71 %.

Nominal and measured concentrations:

- Nominal concentrations: 0.12, 0.38, 1.2, 3.8, and 12 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Four replicate 16 L flow-through glass aquaria (length: 25 cm; width: 18 cm; height: 35 cm) containing approximately 13 L of test medium (height of water level: approx. 29 cm) were used for each test concentration, the control and the solvent control. The glass aquaria were positioned in temperature regulated water baths (two replicates in one water bath). For incubation of the eggs, in each of the replicate aquaria, a stainless steel egg cup (8.5 cm in diameter, 8.0 cm in height) with a stainless steel net bottom was placed. The egg cups were slowly moved up and down by a suitable mechanism to assure a non-turbulent circulation of the test media through the mesh bottom.
- Aeration: The test water was aerated prior to the preparation of the test media.
- Renewal rate of test solution: Into each replicate test vessel, the test medium continuously flowed at a rate of 78 L/day. The volume of the beakers used was 13 L. Thus, the flow rate of the test media through each of the four replicate glass beakers corresponded to a 6-fold theoretical volume exchange per day. Water-flow and dosing systems were calibrated before the start of the dosage. The dosing system was controlled at least once per day and adjusted if necessary. For equilibration of the dosing system, dosing was started 2 days before introduction of the eggs (= test start).
- No. of organisms per vessel: 20 eggs per replicate
- No. of vessels per concentration: Four replicates were used for each treatment.
- No. of vessels per control: Four. Test water without any additions flowed at the same rate into the replicates of the control.
- No. of vessels per vehicle control: Four. For preparation of the solvent control, DMF only was dosed into test water in the same way as described above.
- Biomass loading rate: At the start of the test (Day 0), 80 fertilised eggs were randomly distributed to the replicate test vessels of each treatment. The eggs were handles as carefully as possible using large diameter tubes. The loading rate of the eggs (biomass per volume of test medium in each test vessel) was distinctly lower than 0.5 g/L as requested by the test guideline.
A few days after complete hatching of the larvae, the incubation cups were removed (Day 34 post fertilisation) and the larvae were released into the corresponding 13 L flow through aquaria without exposing the larvae to the air.
The loading rate (based on the volume of the aquaria) of the test fish at the end of the test period was calculated to be a maximum of 1.5 g fish wet weight/L. The loading rate (based on the water flow of 78 L/day per replicate) was calculated to be a maximum of 0.25 g fish wet weight/L/day (this maximum value was calculated at the test nominal concentration of 3.8 mg/L, fulfilling the requirement of the test guideline.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Local tap water (non-chlorinated well water of drinking water quality), reduced for total hardness by ion exchange. The test water was aerated prior to the preparation of the test media.
- Intervals of water quality measurement: Dissolved oxygen concentration, pH, and water temperature were measured in all replicates from all test concentrations and in the controls at the start and end of the test and twice weekly during the test period. Additionally, the water temperature was monitored continuously by a data logger in one control replicate. The total hardness of the test water was measured on 14 dates during the test period. The appearance of the test media was checked each working day. As no visible abnormalities were observed, observations were recorded once a week.

OTHER TEST CONDITIONS
- Photoperiod: Darkness was applied for eggs and larvae until end of hatching Day 29, except for the time when they were inspected. Thereafter, a 16-hour light to 8-hour dark photoperiod with a 30-minute transition period was used.
- Light intensity: Light intensity during the light period was in the range of 150 to 280 Lux.

EFFECT PARAMETERS MEASURED:
Dead eggs, dead larvae or fish were removed at the day of observation. Faeces and surplus food were removed regularly from the test vessels. The test vessels were replaced by clean ones on demand.

- Hatching Rate
The embryonic development and hatching of larvae during hatching period (Day 25 to 29) in the controls and at the test concentrations were recorded for all replicate test vessels at least each working day. Eggs showing a change in colour (white non-transparent colour caused by coagulation and/or precipitation of protein) were considered to be dead. Percent of hatching success was calculated for each replicate by dividing the number of hatched larvae by the number of inserted eggs.

- Development Rate
The development rate was calculated for each replicate during hatching period (Day 25 to Day 29). The mean hatching time represents the mean time span between the start of the hatching period and the hatching of the experimental cohort of larvae. The development rate is the reciprocal of the hatching time (unit: 1/day) and represents that portion of hatching which takes place per day. For the statistical performances the number of hatched larvae observed on inspection Day x were assumed to be hatched at the mean of the time interval between Day x and Day x-1 (1 = length of the inspection interval = 1 day).

- Survival of Larvae and Juvenile Fish
Larvae and juvenile fish were observed for mortality and visible abnormalities at least each working day. The results were recorded at the day of observation. Mortality criteria were absence of respiratory movement and lack of reaction to mechanical stimulus. Dead larvae and fish were removed from the test vessels. The larvae and juvenile fish were observed for visible abnormalities as abnormal appearance (body shape) and behaviour (e.g., uncoordinated swimming, atypical quiescence and atypical behaviour). The survival of the larvae and juvenile fish were observed each day and number of dead larvae and juvenile fish were recorded at the date of observation. The survival rate of the test fish was calculated for each replicate of each test concentration and control by dividing the number of surviving fish by the number of larvae hatched.

- Fish Length and Fish Weight
At the end of the test the test fish were sacrificed and the fish length (from the snout to the end of the caudal fin) and the body wet weight and dry weight (drying for 24 hours at 60 °C) were determined for each individual fish. The mean body length mean wet weight and mean dry weight of the fish were calculated for each replicate per treatment.

- Determination of the NOEC
A statistical evaluation of the biological response data was not performed due to the clear results. As no adverse effect and no concentration-effect relationship was observed at any of the endpoints, the NOEC and LOEC values were determined directly from the raw data.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: The test concentrations were based on a 96-hour LC50 value for rainbow trout which was > 10 mg/L according to information provided by the sponsor. Therefore, the highest test concentration in the definitive test was chosen to be 12 mg/L (higher than the target concentration of 10 mg/L mentioned in the guideline to compensate a possible loss of test material). Furthermore, the correct dosage of the test material at the concentration level chosen was investigated in a flow through dosing pre-experiment and the method applied was shown to be suitable.

RANGE-FINDING STUDY: None.

Reference substance (positive control):

no

Key result

Duration:

89 d

Dose descriptor:

NOEC

Effect conc.:

11.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Effects on eggs, larvae and fish

Details on results:

- Hatching Rate
In the control, the solvent control and at all test concentrations up to and including the highest concentration of 11.2 mg/L (mean measured), first larvae were observed on Day 25 and hatching was completed on Day 29 post fertilisation. The mean hatching success was 87.5 ± 2.9 % in the control and 87.5 ± 6.5 % in the solvent control. Thus, the hatching success in both controls was sufficiently high under the test conditions (the validity limit for hatching success is ≥ 75 %). At all test concentrations the hatching rate was nearly equal to or even slightly higher compared to the solvent control (range: 97 to 104 % of the solvent control value). No concentration-effect relationship was observed.
Therefore, the NOEC for hatching success was determined to be the mean measured concentration of 11.2 mg/L. The LOEC for hatching success could not be determined since it was above the highest concentration tested (> 11.2 mg/L).

- Development Rate of Eggs
In the control and at all test concentrations up to and including 11.2 mg/L (12 mg/L nominal), the development rate of the larvae from fertilisation to hatching was in the range of 91.0 to 118.4 % of the solvent control value. No concentration-effect relationship was observed over the whole concentration range. Therefore, the NOEC for development rate was determined to be the mean measured concentration of 11.2 mg/L. The LOEC for development rate of eggs could not be determined since it was above the highest concentration tested (> 11.2 mg/L).

- Fish Length and Body Weight
For these test parameters the mean values obtained from all test concentrations up to and including 11.2 mg/L were equal to or even slightly higher compared to the solvent control. As no adverse effect was determined, the NOEC for fish body length, fish wet weight and fish dry weight was determined to be 11.2 mg/L. The LOEC for development rate could not be determined since it was above the highest concentration tested (> 11.2 mg/L).

- Survival of Larvae and Juvenile Fish
The mean survival rate of the fish was 87.1 ± 8.8 % in the control and 88.5 ± 7.9 % in the solvent control, demonstrating the suitability of the test conditions (the validity limit for post hatch success is ≥ 75 %). The mean survival rates at the test concentrations up to and including 11.2 mg/L (12 mg/L nominal) were in the range of 82.0 ± 6.4 % to 90.3 ± 2.5 %. These values were nearly identical or even slightly higher compared to the solvent control and no concentration effect relationship was observed. All fish which survived until the end of the test were healthy and showed normal behaviour. Therefore, the NOEC for survival of the embryos and fish was determined to be the mean measured concentration of 11.2 mg/L. The LOEC for survival of larvae and juvenile fish could not be determined since it was above the highest concentration tested (> 11.2 mg/L).

- Analytical Results
The test material concentrations in the freshly prepared and aged application solution used for the dosage of the highest test concentration of nominal 12 mg/L ranged from 93 to 115 % of the nominal value. This shows the correct preparation of the application solution and the stability of the test material in the application solution during its renewal periods of 7 to 8 days.
The analytical measurements from all test concentrations of nominal 0.12 to 12 mg/L during the first seven days of the test (verification of the dosing system) showed recoveries in the range of 81 to 110 % of the nominal values. This shows that the dosage of all test concentration levels used in the test was acceptable.
The test material concentration in the test medium of nominal 12 mg/L (determined as the overall NOEC) varied in the range from 84 to 111 % of the nominal value during the whole exposure period of 89 days (except on Days 14 and 76, where slightly lower values of 74 and 68 % of nominal were determined). The analytical results demonstrate, that the test concentration was sufficiently maintained during the test period of 89 days.
The mean measured test material concentration (calculated as the arithmetic mean of all measurements of the test concentration of 12 mg/L) was calculated to be 11.2 mg/L. The biological results are related to the mean measured test material concentration.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the test material had no toxic effects to rainbow trout up to the highest test concentration of mean measured 11.2 mg/L (NOEC 11.2 mg/L).

Executive summary:

The toxicity of the test material was investigated in an early-life stage toxicity test according to the OECD Guideline 210 and in compliance with GLP.

Freshly fertilised eggs of rainbow trout were exposed to test media containing the test material at nominal concentrations of 0.12, 0.38, 1.2, 3.8 and 12 mg/L under flow-through conditions for the test period of 89 days (60 days post hatch). Additionally, a control and a solvent control (DMF) were tested in parallel. 

At the start of the test, 80 eggs each (divided into 4 replicates) were distributed to the test concentrations, control and solvent control. The eggs, larvae and juvenile fish were observed for toxic effects on their development, growth and survival.

The test material concentration in the test medium of nominal 12 mg/L (determined as the overall NOEC) varied in the range from 84 to 111 % of the nominal value during the whole exposure period of 89 days (except on Days 14 and 76, where slightly lower values of 74 and 68 % of nominal were determined). The analytical results demonstrate that the test concentration was sufficiently maintained during the test period of 89 days. The mean measured test material concentration (calculated as the arithmetic mean of all measurements of the test concentration of 12 mg/L) was calculated to be 11.2 mg/L. The NOEC and LOEC were related to the mean measured test material concentrations of the test material.

Hatching rate, development rate of embryos, survival of larvae and juvenile fish, fish body length and fish wet weight and dry weight all resulted in a NOEC of 11.2 mg/L and a LOEC of > 11.2 mg/L.

The NOEC values for each of the test parameters assessed, the overall NOEC of the test material for early life stages of rainbow trout was determined to be the mean measured test concentration of 11.2 mg/L (nominal 12 mg/L), since no toxic effect on the eggs, larvae or fish was observed up to and including this test concentration. The overall LOEC could not be determined due to the absence of toxic effect of the test material up to the highest test concentration (LOEC > 11.2 mg/L).

Under the conditions of the study, the test material had no toxic effects to rainbow trout up to the highest test concentration of mean measured 11.2 mg/L.

Reference 2

Endpoint:

long-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

14 April 1993 to 12 May 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 204 (Fish, Prolonged Toxicity Test: 14-day Study)

Version / remarks:

prolonged to 28 days

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Concentration controls: Samples were taken on day zero and then at weekly intervals from the test aquaria and were analysed for content of test material.
Test material stability: The stability of the test material in water was verified by the analytical concentration control. The samples for the weekly concentration controls during the study had generally always been taken out of the aquaria immediately before the stock dilutions in the storage vessels were replaced except at the beginning of the study.
Storage Conditions until Analysis: Refrigerator

Vehicle:

yes

Remarks:

NaOH

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Since the acid is hardly soluble in water, a concentrated stock solution was prepared by dissolving the aliquot amount of the test material in water by adding an aliquot quantity of NaOH (about 10 %). This mixture was then stirred on a magnetic stirrer until the test material was completely dissolved. Thus, the water-soluble sodium salt was prepared. This concentrated stock solution was then used to prepare all stock solutions for the test groups. This procedure to dissolve the test material is judged to be acceptable since, due to the test water which has a pH of 8.3 - 8.5, in the test water of the aquaria finally a salt would be formed.
Preparation of the first batch of the stock dilutions on day -1; replacement of the stock dilutions in the storage vessels on days 7, 14 and 21.
The homogeneity is guaranteed since the stock dilutions are true solutions.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout
- Strain: Walbaum 1792
- Age at study initiation (mean and range, SD):
- Length at study initiation: 6.0 cm (5.4 - 6.7)
- Weight at study initiation: 1.9 g (1.5 - 2.5). Eight days prior to the beginning of the study a batch of test fish of approximately uniform weight was established by weighing the fish individually. Then a weight range of 2.0 g ± 0.5 was fixed. On the day the study was started the fish were weighed again individually. Only individuals that were in the fixed weight range were used; the others were discarded. The fish were then allocated to the test groups by following a randomisation plan of the laboratory.
- Feeding during test: During the study about 4 % of their mean body weight on workdays daily in 2 portions and on non-working days in one portion. Beginning with week 2 of the study the amount of feed was generally increased by 25 % each week (calculated for the initial weight in each case and the number of survivors if applicable).
- Food type: The animals received "Ssniff" trout starter FB-50/size 1, of "Ssniff", Soest, FRG.
- Corpulence factor: 0.9. The corpulence factor K is calculated from the weight W (g) and the length L (cm) measured from the tip of the mouth to the distal end of the caudal fin according to the formula:

K = (100 x W) / L^3

- Sex ratio: At that juvenile age the sex cannot be reliably determined by a visual inspection. Therefore the animals were used in an unspecified sex ratio.

ACCLIMATION
- Acclimation period: The total period of adaptation to the test conditions, including approximate test temperature, was about three months.
- Acclimation conditions: The fish were housed during adaptation under conditions similar to those in the study. The fish were adapted to the test water, the approximate test temperature and feed from their arrival in the laboratory until the beginning of the study. During the adaptation a sufficient number of test fish were kept in plastic aquaria with a constant volume of about 230 or 310 L in a continuous flow-through system with aeration. The photoperiod for the total adaptation period was 16 hours light and 8 hours darkness.
- Type and amount of food: The animals received "Ssniff" trout starter FB-50/size 1, of "Ssniff", Soest, FRG.
- Feeding frequency during acclimation: During adaptation about 1 to 2 % of their mean body weight daily in 2 portions on workdays and in one portion on non-working days; additionally they received on workdays artemia during the initial 2 weeks.
- Health during acclimation: Mortality during final adaptation period (about 1 week) of the batch established for the study (200 fish) was zero. Soon after arrival in the laboratory the fish were treated prophylactically twice with 0.05 mg/L malachite green chloride and once with 10 mg/L tetracycline hydrochloride for about 24 hours each. Immediately before the fish were placed into the test aquaria they were inspected for health. Only healthy fish of normal appearance were used.

Residues in the diet: The feed used in this type of studies is assayed at regular intervals for contaminants. In view of the aim and duration of this study, the contaminants contained in commercial feed should have no influence on the results.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Remarks on exposure duration:

Since delayed compound-related effects could not be excluded the study was extended beyond the minimum duration of 14 days in accordance with the recommendation of the guideline to a period of 4 weeks, the maximum duration of that type of study.

Hardness:

2.3 mmol/L

Test temperature:

16 °C

pH:

8.3 - 8.5

Dissolved oxygen:

9 to 10 mg/L which is roughly about 90 to 100 % of the maximum saturation.

Nominal and measured concentrations:

Nominal: 0, 1, 10, 50 and 100 mg/L
The measured concentrations averaged 96 % of the range 88.8 - 105.1 %

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquaria
- Size of vessel: The test vessels were 60 x 35 x 40 cm.
- Material, size, headspace, fill volume: Glass in a stainless steel frame with an over flow. The aquaria were adapted to maintain a constant water volume in each test aquarium of about 60 L. The construction materials that came into contact with the water and the dilution of the test substance did not contain any significant amounts of substances that could be washed out and did not absorb any substantial amounts of the test material. Plastics were only used where absolutely necessary. Most of the apparatus consisted of glass. The tubes through which the stock solution was metered into the mixing tanks were made of Teflon. The apparatus was checked daily for proper function.
- Aeration: The aquaria were aerated continuously with oil-free air.
- Renewal rate of test solution: The flow rates of the test solutions were 10 L/hour/test aquarium. The flow meter (rotameter) level was checked daily and corrected if necessary.
- No. of organisms per vessel: 20 fish per test group.
- No. of vessels per concentration: One
- No. of vessels per control: One

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Drinking water from the city of Frankenthal, purified over active carbon and aerated (fish laboratory quality). The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and the Technical Services of BASF Aktiengesellschaft as well as for presence of microbes by a contract laboratory. On the basis of the findings the study director decided whether the level of contaminants is acceptable. The German Drinking Water Regulation (Trinkwasserverordnung, Bundesgesetzblatt, May 22, 1986) serves as a guideline for maximum tolerable contaminants.
- Intervals of water quality measurement:
Test temperature: Measured daily
pH: pH 91, WTW, Weilheim, FRG, with electrode: pH EinstabmeBkette (single-rod measuring device). These measurements were generally performed twice weekly.
O2 measurement: Oxi 91 and electrode: EO 90 WTW, Weilheim, FRG. These measurements were generally performed twice weekly.
Total hardness: The total hardness was determined generally in weekly intervals by titration with EDTA.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light/8 hours darkness.

EFFECT PARAMETERS MEASURED: Mortality was determined daily. Clinical signs were assessed at least on each work day. The individual body weights and the individual body lengths (from the tip of the snout to the distal end of the caudal fin) were determined at the beginning (day 0) and at the end of the study (day 28). About 16 hours before the determination of the body weight the feed was withdrawn.

RANGE-FINDING STUDY : No
- The results of a preceding acute static study on the rainbow trout served as the basis for selecting the concentrations. The following results were obtained (96-hour values): The highest concentration without mortality (LC0) was 147 mg/L; the NOEC was 68.1 mg/L. Since it was assumed that toxic effects could occur at lower concentrations during the 28-day test, the concentration levels were selected in such a way that the threshold level for lethal and other visible effects and the "No Observed Effect Concentration (NOEC)" should be attained.

Reference substance (positive control):

not required

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

Mortality: In the control group as well as in all groups containing test substance (1, 10, 50 and 100 mg/L) no mortalities occurred.
The lowest concentration at which mortalities occurred was greater than 100 mg/L.
The threshold level of lethal effects was greater than 100 mg/L.
The highest concentration tested without mortalities was 100 mg/L.

Toxic signs and symptoms: No marked toxic signs were observed in all groups that contained test compound. Only in the highest test group (100 mg/L) a slight discolouration was seen on days 2 and 5 of the study.
The threshold level for toxic signs was about 100 mg/L.
The "No Observed Effect Concentration (NOEC)" for toxic signs was 50 mg/L.

Body weight: At the beginning (day 0) the mean body weights for all groups including control ranged from 1.9 - 2.0 g. At the end of the study (day 28) the mean body weight of the control group was 6.6 g and those of the concentration groups 1, 10, 50 and 100 mg/L were 6.5; 6.7; 6.4 and 5.9 g which is an increase of about factors 2.9 to 3.5 compared to the initial weight. The weight gain in the highest test group was statistically significantly reduced at the p ≤ 0.05 level compared to the control group.
Thus, under the conditions of this study, the no effect concentration for the impairment of the body weight was 50 mg/L.

Body length: At the beginning of the study (day 0) the mean body length for all groups including the control group ranged from 6.0 - 6.1 cm. At the end of the study (day 28), the mean body length of the control group was 8.6 cm and those of the test groups with the concentrations 1, 10, 50 and 100 mg/L were 8.6, 8.7, 8.4 and 8.0 cm which is an increase of about factors 1.3 to 1.4 compared to the initial length. The gain in body length in the highest test group was statistically significantly reduced at the p ≤ 0.01 level compared to the control group.
Thus, under the conditions of this study, the no effect concentration for the impairment of the body length was 50 mg/L.

Reported statistics and error estimates:

The statistical evaluation of the body weight and the body length at the beginning (day 0) and at the end of the study was performed using Dunnett's test.

Validity criteria fulfilled:

not specified

Remarks:

expected to be valid source

Conclusions:

Under the conditions of this study the overall NOEC was determined to be 50 mg/L for the rainbow trout (nominal concentration).
The threshold level for lethal effects was greater than 100 mg/L, for toxic signs about 100 mg/L and for the growth parameters body weight plus length greater than 50 and smaller than 100 mg/L (nominal concentrations).

Executive summary:

The toxicity of the test material was assessed according to OECD Test Guideline 204 and in compliance with GLP

The test material was tested for its sublethal toxic effects on rainbow trout, 20 fish/test group, in the concentrations 0 (control), l, 10, 50 and 100 mg/L in a flow-through system for a period of 4 weeks, and the following results were obtained:

Mortality:

- In the control group as well as in all groups containing test compound (l, 10, 50 and 100 mg/L) no mortalities occurred.

- The threshold level of lethal effects was greater than 100 mg/L. Highest concentration tested without test material-related mortalities was 100 mg/L.

Toxic signs (symptoms): No marked toxic signs were seen in all test groups. Only in the highest dose group (100 mg/L) a slight discolouration was seen on days 2 and 5 of the study. The threshold level for toxic signs was about 100 mg/L.

The "No Observed Effect Concentration" for toxic signs was 50 mg/L.

Body weight and length: Compared with the control group, at the end of the study, the mean body weight in the test group 100 mg/L was statistically significantly smaller at the p ≤ 0.05 level. The mean body length at the end of the study was statistically significantly smaller in this test group at the p ≤ 0.01 level.

Under the conditions of this study the threshold level for the growth parameters body weight and length was greater than 50 and smaller than 100 mg/L and the no effect concentration for these parameters was 50 mg/L.

Under the conditions of this study the overall NOEC was determined to be 50 mg/L for the rainbow trout (nominal concentration).

The threshold level for lethal effects was greater than 100 mg/L, for toxic signs about 100 mg/L and for the growth parameters body weight plus length greater than 50 and smaller than 100 mg/L (nominal concentrations).

Description of key information

Peither (2015)

Under the conditions of the study, the test material had no toxic effects to rainbow trout up to the highest test concentration of mean measured 11.2 mg/L.

Supporting Study: Munk (1993)

Under the conditions of this study the overall NOEC was determined to be 50 mg/L for the rainbow trout (nominal concentration).

The threshold level for lethal effects was greater than 100 mg/L, for toxic signs about 100 mg/L and for the growth parameters body weight plus length greater than 50 and smaller than 100 mg/L (nominal concentrations).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

11.2 mg/L

Additional information

Peither (2015)

The toxicity of the test material was investigated in an early-life stage toxicity test according to the OECD Guideline 210 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Freshly fertilised eggs of rainbow trout were exposed to test media containing the test material at nominal concentrations of 0.12, 0.38, 1.2, 3.8 and 12 mg/L under flow-through conditions for the test period of 89 days (60 days post hatch). Additionally, a control and a solvent control (DMF) were tested in parallel. 

At the start of the test, 80 eggs each (divided into 4 replicates) were distributed to the test concentrations, control and solvent control. The eggs, larvae and juvenile fish were observed for toxic effects on their development, growth and survival.

The test material concentration in the test medium of nominal 12 mg/L (determined as the overall NOEC) varied in the range from 84 to 111 % of the nominal value during the whole exposure period of 89 days (except on Days 14 and 76, where slightly lower values of 74 and 68 % of nominal were determined). The analytical results demonstrate that the test concentration was sufficiently maintained during the test period of 89 days. The mean measured test material concentration (calculated as the arithmetic mean of all measurements of the test concentration of 12 mg/L) was calculated to be 11.2 mg/L. The NOEC and LOEC were related to the mean measured test material concentrations of the test material.

Hatching rate, development rate of embryos, survival of larvae and juvenile fish, fish body length and fish wet weight and dry weight all resulted in a NOEC of 11.2 mg/L and a LOEC of > 11.2 mg/L.

The NOEC values for each of the test parameters assessed, the overall NOEC of the test material for early life stages of rainbow trout was determined to be the mean measured test concentration of 11.2 mg/L (nominal 12 mg/L), since no toxic effect on the eggs, larvae or fish was observed up to and including this test concentration. The overall LOEC could not be determined due to the absence of toxic effect of the test material up to the highest test concentration (LOEC > 11.2 mg/L).

Under the conditions of the study, the test material had no toxic effects to rainbow trout up to the highest test concentration of mean measured 11.2 mg/L.

Supporting Study: Munk (1993)

The toxicity of the test material was assessed according to OECD Test Guideline 204 and in compliance with GLP. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was tested for its sublethal toxic effects on rainbow trout, 20 fish/test group, in the concentrations 0 (control), l, 10, 50 and 100 mg/L in a flow-through system for a period of 4 weeks, and the following results were obtained:

Mortality:

- In the control group as well as in all groups containing test compound (l, 10, 50 and 100 mg/L) no mortalities occurred.

- The threshold level of lethal effects was greater than 100 mg/L. Highest concentration tested without test material-related mortalities was 100 mg/L.

Toxic signs (symptoms): No marked toxic signs were seen in all test groups. Only in the highest dose group (100 mg/L) a slight discolouration was seen on days 2 and 5 of the study. The threshold level for toxic signs was about 100 mg/L.

The "No Observed Effect Concentration" for toxic signs was 50 mg/L.

Body weight and length: Compared with the control group, at the end of the study, the mean body weight in the test group 100 mg/L was statistically significantly smaller at the p≤0.05 level. The mean body length at the end of the study was statistically significantly smaller in this test group at the p≤0.01 level.

Under the conditions of this study the threshold level for the growth parameters body weight and length was greater than 50 and smaller than 100 mg/L and the no effect concentration for these parameters was 50 mg/L.

Under the conditions of this study the overall NOEC was determined to be 50 mg/L for the rainbow trout (nominal concentration).

The threshold level for lethal effects was greater than 100 mg/L, for toxic signs about 100 mg/L and for the growth parameters body weight plus length greater than 50 and smaller than 100 mg/L (nominal concentrations).