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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Chemical name: Phosphinic acid, phenyl(2,4,6-trimethylbenzoyl)-, ethyl ester
BMG sample no.: M1302-00840-01
CAS no.: 84434-11-7
Empirical formula: C18H21O3P
Physical form: Liquid, viscous
Water solubility: Insoluble at 20°C: 35 mg/l
Purity (1H-NMR): 95.7 g/100 g
Purity (GC): 97.0 area-% (DB-1) – 97.8 area-% (DB-1701)
Test substance storage: At room temperature, protected from light
Analytical monitoring:
yes
Details on sampling:
HPLC analyses of the test substance concentrations were conducted at the beginning, after 24 h (old and renewed solutions), 48 h (old and renewed solutions), 72 h (old and renewed solutions) and after 96 h of exposure.
Vehicle:
no
Details on test solutions:
Lucirin TPO-L is only partially soluble. Moreover, Lucirin TPO-L is not stable in the aquatic environment. Therefore, 150 mg Lucirin TPO-L was directly added to natural water. After a stirring duration of 3 h, the suspension was filtered (glass fibre filter) and analyzed by HPLC. The resulting concentration was diluted to give the following nominal concentrations of Lucirin TPO-L as defined by a range finding test: 0.25,
0.5, 1.0, 2.0, 4.0 and 8.0 mg/l.
Semi-static conditions were applied. Fresh test solutions were prepared after 24, 48 and 72 h. The fish were transferred into these fresh test solutions after 24, 48 and 72 h of exposure.
A blank consisting of dilution water only, without any addition of test substance or other additives, served as a control.
At least 1 h before the start of the exposure, the test vessels were filled with the test solutions to allow equilibration of pH, oxygen and temperature. The pH was not adjusted.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Test organism: Danio rerio (Zebrafish)
Culturing: Population of parental and young fish are held in 40 l glass aquaria. The bottom of the aquaria is covered with gravel (≤1.5 cm).
Illumination: 16 h a day
Temperature: 21-25 °C, controlled by a thermometer.
Medium: Continuously aerated natural water of the town of Schlieren.
Feed: The fish are fed commercially prepared diet Sera micropan and Sera vipan (Sera apanristic GmbH, D-52525 Heinsberg, Germany).
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
Total Hardness
°f H: 16.1 - 20.9
mmol/l: 1.61 - 2.09
mg/l: 161 - 209

CO3 – Hardness
°f H: 14.6 - 18.8
mg/l: 146 - 188

Rest – Hardness
°f H: 1.5 - 2.1
mg/l: 15 - 21
Test temperature:
21 - 22 °C
pH:
6.5 - 8.5

The pH values were in the recommended range of 6.0–8.5.
Dissolved oxygen:
7.1 - 8.1 mg/L
Nominal and measured concentrations:
Chemical analyses revealed that the concentrations were reduced by a factor of 2 to 3 within each 24 h period. Geometric mean concentrations were calculated for each 24 h period. The arithmetic means (AM0-96h) of the geometric mean concentrations over each 24 h period for the nominal concentrations of 0.5, 1.0, 2.0, 4.0 and 8.0 mg/l were 0.162, 0.349, 0.740, 1.29, 2.50 and 5.09 mg/l, respectively
Details on test conditions:
Test vessel: Glass aquaria of 2 l (14.5 x 14.0 x 9.5 cm) filled with 1.0 liter of water
Test medium: Continuously aerated tap water of the town of Schlieren
Oxygen concentration (target): The total dissolved oxygen concentration is kept >60% of the saturation value with aeration via Pasteur pipettes
Number of fish: 7 fish per test concentration and control (target: total length of 1-3 cm).
Light: 16 h photoperiod a day, supplied by overhead white fluorescent tubes, no transition period
Temperature (target): 21-25 °C
Test type: Semi-static exposure conditions
Test duration: 96 h

Handling
The exposure began, when all the fish were randomly placed in the test vessels. The transfer was performed in an impartial manner to eliminate bias. Fine-mesh dip net was used to transfer fish, taking care to minimise possible stress due to handling. Fish which were damaged or dropped during the transfer were not used. Transfer by fine-mesh dip net to fresh media was performed every 24 h.

Testing approach and test concentrations:
Limit test (1st test series) under static conditions:
A 1st definitive test was performed as a limit test at the loading rate of 5.64 mg/l under static conditions. The selected loading was based on the results of the preceding GLP-tests with algae (in the course of study no. A13-00197) and Daphnia (in the course of study no. A13-00198) yielding EC50 values of 12.6 mg/l and 5.64 mg/l loading rates, respectively. At the loading rate of 5.64 mg/l, 3 of a total of 7 fish died after 48 h of exposure. No mortality was observed in the blank control.

2nd test series under static conditions:
Based on the results of the 1st test series, a 2nd definitive test was performed as a full test with 5 loading rates under static conditions. The selected loading rates were 1.6, 2.4, 3.5, 5.3 and 8.0 mg/l. Based on the results of this 2nd definitive test, another full test with 5 loading rates was performed. The selected loading rates were 0.10, 0.32, 1.0, 3.2 and 10 mg/l.

3rd test series under static conditions
Based on the results of this 2nd definitive test, another full test with 5 loading rates was performed under static conditions. The selected loading rates were 0.10, 0.32, 1.00, 3.20 and 10 mg/l. The stirring duration was 72 h. Low concentrations, lower than in the preceding tests, were measured (results not shown). It was assumed that the test material is not stable. Therefore, non-GLP stirring pretests were performed prior to further fish tests to evaluate the appropriate test procedure for Lucirin TPO-L.

Non-GLP stirring pretests
Several non-GLP pretests to find the optimal procedure of stirring at loading rates of 50, 100 and 150 mg/l and different stirring durations were performed. The results were as follows:
Concentration of Lucirin TPO-L in a 1st non-GLP pretest with loading rates of 50, 100 und 150 mg/l in natural water and a stirring duration of 72 h:
• 50 mg/l: 2.7 mg/l
• 100 mg/l: 4.1 mg/l
• 150 mg/l: 31 mg/l
Concentration of Lucirin TPO-L in a 2nd non-GLP pretest with loading rates of 50, 100 und 150 mg/l in natural water and a stirring duration of 168 h:
• 50 mg/l: 0.17 mg/l
• 100 mg/l: 1.8 mg/l
• 150 mg/l: 8.9 mg/l
Concentration of Lucirin TPO-L in a 3rd non-GLP pretest with loading rates of 50, 100 und 150 mg/l in natural water and a stirring duration of 24 h:
• 50 mg/l: 13.5 mg/l
• 100 mg/l: 37.4 mg/l
• 150 mg/l: 18.5 mg/l
Concentration of Lucirin TPO-L in a 4th non-GLP pretest with loading rates of 50, 100 und 150 mg/l in natural water and again a stirring duration of 24 h:
• 50 mg/l: 12.3 mg/l
• 100 mg/l: 7.2 mg/l
• 150 mg/l: 37.2 mg/l

The results suggest that the test material is not stable. The lower the concentration, the higher the area of an additional peak in the chromatogram. Therefore, it was decided to perform a 5th pretest by adding 50, 100 and 150 mg/l directly to natural water. The concentrations of Lucirin TPO-L were frequently analyzed as shown in the table below:

Based on the results of the last non-GLP pre-test, it was decided to run the fish study as follows:
• 150 mg Lucirin TPO-L is directly added to natural water. After a stirring duration of 3 h, the suspension is filtered and analyzed by HPLC. The resulting concentration is diluted to give nominal concentrations of Lucirin TPO-L of 0.1, 1.0 and 10 mg/l for a range finding test with 3 fish per concentration.
• Semi-static conditions are applied. Fresh test solutions are prepared after 24, 48 and 72 h. The fish are transferred into these fresh test solutions after 24, 48 and 72 h of exposure.

Pretest under semi-static conditions:
The pretest under semi-static conditions and nominal concentrations of 0.1, 1.0 and 10 mg/l and 3 fish per concentration resulted in the cumulative mortalities presented in the table below:
Based on these results, the full test under semi-static conditions was performed with the following six nominal concentrations (spacing factor: 2): 0.25, 0.50, 1.0, 2.0, 4.0 and 8.0 mg/l.

Observations and measurements:
Oxygen, pH, temperature:
Measurements were made prior to the addition of the fish to the test solutions and after 2, 24, 48, 72 and 96 h of exposure

Observations:
Observations of the fish were made after 2, 24, 48, 72 and 96 h of exposure. Mortality of the fish was recorded at the same time intervals. Dead fish were removed immediately from the test solution and weight and length were recorded.

Water analysis:
The parameters analyzed by the Kantonales Labor Zürich are based on the values obtained from three groundwater wells (aquifer of the river Limmat). These three wells represent the major fraction (90%) of the drinking water of the town of Schlieren. The remaining 10% derive from spring water. The water quality fulfills all legal requirements. Therefore, no chlorination is needed and the water can be used in the tests without any treatment.

Evaluation of the test results:
Definition of mortality/morbidity and median lethal concentration:
Fish are considered to be dead if they show no reaction after touching the caudal fin and there is no visible movement (e.g. gill movements).
Fish are considered to be moribund (in the state of dying) if they exhibit serious sub-lethal effects (e.g. lying immobile at the bottom of the tank while still reacting to touching). Fish with these symptoms are likely to be compromised and hardly ever recover. In such a case, it is decided to humanely kill the fish in accordance with animal welfare. Moribund fish are considered to be dead with respect to the calculation of the LC50 value. The median lethal concentration (LC50 value) is the concentration killing 50% of the fish after a certain period of exposure.

Calculation of the LC50 value:
The LC50 incl. 95% confidence limits was calculated with ToxRat® Standard Version 2.10 (ToxRat® Solutions GmbH, Alsdorf, Germany) using moving average computation after Thompson2.

Determination of no observed effect concentration:
The no observed effect concentration (NOEC) is the highest test concentration for which the test item is observed to have no biologically significant effects when compared to the controls. Mortality rates of ≤10% (1 fish) are regarded as not biologically significant.

Validity criterion:
The mortality in the control should not exceed 10% (or 1 fish, if less than 10 fish are used) at the end of the test.

Data compilation:
The following data were recorded on data sheets and transcribed for compilation and analysis after 2, 24, 48, 72 and 96 h: amount of test material applied, number of dead fish or abnormal responses of the fish, weight and length of all fish (fish of control(s) and of test concentration(s)), pH, O2 and temperature. The following data were recorded on-line (or on data sheets, as appropriate): HPLC measurements.
The HPLC is validated with respect to data collection, storage and retrievability. Statistical analysis was performed with respect to regression data.
Reference substance (positive control):
not specified
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.89 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% confidence limits: 1.46–2.43 mg/l
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 1.29 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
Results and discussion:
Mortality and other symptoms:
Incidences of mortality are presented in Table 2. Figure 1 shows the mortality observed in the test graphically. Specification of symptoms observed on affected fish and data of dead and surviving fish are listed in Table 3 and 4. At the nominal concentration of 8.0 mg/l, all seven fish were declared as moribund (in the state of dying) after 24 h. At 4.0 mg/l, all seven fish were dead after 48 h of exposure. One fish died at 2.0 mg/l after 72 h of exposure. No mortality was observed at 1.0, 0.50, 0.25 mg/l and in the blank control within 96 h. Toxic effects other than mortality or moribund state were observed at the nominal concentration of 8.0 mg/l after 2 h of exposure. All seven fish were swimming on the back. Further effects were observed at 4.0 mg/l after 24 h (loss of coordination) and at 0.25 mg/l after 48 h (one fish swimming on the back) (Table 3).

Estimation of LC0, LC50 and LC100:
The effective concentrations (NOEC, LC50, LC100) of Lucirin TPO-L were assessed based on the arithmetic means of the geometric mean concentrations per 24 h period (AM0-96h). The lethal concentration (LC50) of Lucirin TPO-L to Danio rerio (Zebrafish) was calculated to be 1.89 mg/l (95% confidence limits: 1.46–2.43 mg/l). The no-observed-effect concentration (NOEC) was 1.29 mg/l since the one fish that died after 72 h is considered not to represent a significant effect as defined by the validity criteria of the test (i.e. test is regarded as valid with mortality of 1 fish in control). 100% mortality (LC100) was observed at 2.50 mg/l after 48 h of exposure.

pH-values and temperature:
pH-values, oxygen concentrations and temperature during the definitive test are presented in Tables 5, 6 and 7.

Test concentrations:
Chemical analyses revealed that the concentrations were reduced by a factor of 2 to 3 within each 24 h period. Geometric mean concentrations were calculated for each 24 h period. The arithmetic means (AM0-96h) of the geometric mean concentrations over each 24 h period for the nominal concentrations of 0.5, 1.0, 2.0, 4.0 and 8.0 mg/l were 0.162, 0.349, 0.740, 1.29, 2.50 and 5.09 mg/l, respectively (Table 8, Table 9).
Chromatograms of the nominal concentration of 8.0 mg/l at time 0 and 24 h are presented in Figure 2 and Figure 3.

Validity of the test
The validity criterion (mortality in the control should not exceed 10%) was fulfilled.

Table 2 Cumulative mortality

Nominal concentration (mg/l) Cumulative number of dead fish observed at different exposure times
2h 24h 48h 72h 96h
Control 0 0 0 0 0 0
0.25 0 0 0 0 0
0.5 0 0 0 0 0
1 0 0 0 0 0
2 0 0 0 1 1
4 0 1 7 b) b)
8 0 7 a) b) b)

a) All 7 fish were swimming on the back after 2 h of exposure and were immobile at the bottom of the test vessel after 24 h of exposure while still reacting to touching. For this reason, they were declared as moribund.  

b) All fish died, no further observations were made.

Table 3 Specification of symptoms observed on affected fish and data of dead fish

Nominal concentration (mg/l) Number of affected fish a)  Time of score (h) Total body length (mm)  Wet body weight (mg) Symptom b)
8  7/7 2 - - swimming on the back
8 7/0  24 20 59 immobile at the bottom of the test vessel showing slight reaction upon touching (moribund state) 
12 27
24 95
26 130
19 62
16 36
20 72
4  1/6 24 15 43 dead, at bottom
4  6/6 24 - -  loss of coordination
4  6/0 48 22 113 dead, at bottom
17 66
14 30
13 22
16 59
14 24
0.25  1/7 48 - - swimming on the back
2  1/6 72 17 34 dead, at bottom

a) Number of affected fish / number of surviving fish.

b) E.g. hyper-/hypoactivity, immobility, swimming at the bottom/surface, darkened pigmentation / discoloring.

Table 4 Total length and wet weight of fish

Nominal concentration (mg/l)   Mean values
Control 0 length (mm) 20 19 20 16 18 22 20 19
weight (mg) 81 56 71 41 48 97 61 65
0.25 length (mm) 16 12 16 15 17 12 12 14
weight (mg) 28 17 37 26 30 17 18 25
0.5 length (mm) 14 14 16 20 18 20 12 16
weight (mg) 18 20 35 73 57 63 17 40
1 length (mm) 18 16 16 14 15 16 18 16
weight (mg) 54 26 32 18 21 33 32 31
2 length (mm) 19 16 20 17 19 16 17 18
weight (mg) 48 25 47 35 39 18 34 35
4 length (mm) 15 22 17 14 13 16 14 16
weight (mg) 43 113 66 30 22 59 24 51
8 length (mm) 20 12 24 26 19 16 20 20
weight (mg) 59 27 95 130 62 36 72 69

Values in italics are for fish that were not surviving after 96 h of exposure (see also Table 3).

Fish were of the recommended total length of 10-30 cm.

Validity criteria fulfilled:
yes
Remarks:
The validity criterion (mortality in the control should not exceed 10%) was fulfilled.
Conclusions:
The 96 hour EC50 of the test material to fish was determined to be 1.89 mg/l under the conditions of the test.
Executive summary:

The acute lethal toxicity of Lucirin TPO-L, to Danio rerio (Zebrafish) was investigated under static exposure conditions over a period of 96 h.

Lucirin TPO-L is only partially soluble. Moreover, Lucirin TPO-L is not stable in the aquatic environment. Therefore, 150 mg Lucirin TPO-L was directly added to natural water. After a stirring duration of 3 h, the suspension was filtered (glass fibre filter) and analyzed by HPLC. The resulting concentration was diluted to give the following nominal concentrations of Lucirin TPO-L as defined by a range finding test: 0.25,

0.5, 1.0, 2.0, 4.0 and 8.0 mg/l. Semi-static exposure conditions were applied. Fresh test solutions were prepared after 24, 48 and 72 h. The fish were transferred into these fresh test solutions after 24, 48 and 72 h of exposure.

Chemical analyses revealed that the concentrations were reduced by a factor of 2 to 3 within each 24 h period. Geometric mean concentrations were calculated for each 24 h period. The arithmetic means (AM0-96h) of these geometric mean concentrations for the nominal concentrations of 0.5, 1.0, 2.0, 4.0 and 8.0 mg/l were 0.162, 0.349, 0.740, 1.29, 2.50 and 5.09 mg/l, respectively.  

At the nominal concentration of 8.0 mg/l, all seven fish were declared as moribund (in the state of dying) after 24 h exposure. At 4.0 mg/l, all seven fish were dead after 48 h of exposure. One fish died at 2.0 mg/l after 72 h of exposure. No mortality was observed at 1.0, 0.50, 0.25 mg/l and in the blank control within 96 h.

Toxic effects other than mortality or moribund state were observed at the nominal concentration of 8.0 mg/l after 2 h of exposure. All seven fish were swimming on the back. Further effects were observed at 4.0 mg/l after 24 h (loss of coordination) and at 0.25 mg/l after 48 h (one fish swimming on the back).

Based on these data, the lethal concentration (LC50) of Lucirin TPO-L to Danio rerio (Zebrafish) was calculated to be 1.89 mg/l (95% confidence limits: 1.46–2.43 mg/l).  

The no-observed-effect concentration (NOEC) was 1.29 mg/l.

100% mortality (LC100) was observed at 2.50 mg/l after 48 h of exposure.  

The effective concentrations (NOEC, LC50, LC100) of Lucirin TPO-L are based on the arithmetic means of the geometric mean concentrations per 24 h period (AM0-96h).

Description of key information

The study was conducted to recognised testing guidelines, and with GLP certification.

Key value for chemical safety assessment

LC50 for freshwater fish:
1.89 mg/L

Additional information