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Long-term toxicity to aquatic invertebrates

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long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
The test was performed according to an accepted international guideline according to the principles of GLP. The results of this test are considered to be reliable with restriction as the exposure concentrations were not maintained within an acceptable range at the end of the test. However, as fecundity at the NOEC (0.254 mg/l) is comparable to fecundity at 0.831 mg/l, the results are considered to be robust and the decrease in the measured levels at the end of the test is not considered to have had a major impact on the test result
according to guideline
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
Specific details on test material used for the study:
Purity 99.9%, lot no. GG01-DK
Analytical monitoring:
Details on sampling:
The test substance was kept in the refrigerator of the laboratory.
0.75 mL of each test solution at the beginning of exposure and at the end of exposure was collected in a measuring vial, and an equal amount of acetonitrile was added and analyzed by HPLC.
HCO-60:dimethylformamide 1:1 (v/v)
Details on test solutions:
The test media were prepared from a concentrated stock of the test item made in a mixture of HCO-60 and dimethylformamide (DMF) 50:50 v/v. As an auxilliary agent was used to prepare the test media, the study employed two control groups. In one of these groups, Daphnia were exposed to dilution medium (Elendt M4) alone. In the second control group, Daphnia were exposed to dilution medium containing the maximum concentration (80 mg/L) of the solvent mixture used to prepare the test media. The maximum concentration of the auxilliary mixture in the control and test groups is within the recommended limits of the test guideline (0.1 mL/L, eqivalent to 100 mg/L).
Daphnia were exposed at nominal concentrations of 0.025, 0.08, 0.25, 0.8 and 2.5 mg/L. The range of concentrations for the definitive test was based on the results of an acute (48-h) test with the same substance (EC50 2.41 mg/L).
Test organisms (species):
Daphnia magna
Details on test organisms:
Juvenile (<24 hours old) Daphnia magna were exposed for 21 days to triphenyl phosphate prepared in Elendt M4.

- Source: Environment Agency National Institute for Environmental Studies
- Date of receipt: 1995-07-18
- Management after acquisition: Subculture (maximum breeding period: 4 weeks, changing frequency; at least 3 times per week)

- Parental acclimation: Acclimatization period; January 12, 2000 - February 3, 2000; Parent's mortality rate is less than 5% for two weeks before the start of exposure, and generation of diapausing eggs and males is recognized
- breeding water: Dilution water (Elendt M4)
- breeding density: 1 head / 80 mL breeding water (25 heads / 2 L)
- Water temperature: 20 ± 1 ° C
- Lighting: Indoor light, 16 hours light (less than 800 lux) / 8 hours dark
- Bait: Chlorella vulgaris
- Feeding amount: 0.2 mg C (Organic carbon content) per Daphnia / day
- Confirmation of sensitivity: Testing 48 h with potassium dichromate (special grade reagent): EC50 = 0.57 mg/L, value is consistent with the range typically found at the test facility since 1998 (0.59-1.02 mg/L, n=3).
Test type:
Water media type:
Limit test:
Total exposure duration:
21 d
210-240 mg/l CaCO3
Test temperature:
7-3 -8.8
Dissolved oxygen:
7.2 -8.9
Nominal and measured concentrations:
The measured levels of the test item ranged from 96 to 111 % of the nominal concentrations from Day 0 to Day 12 however, the measured levels in the expired (48 hours old) medium at the lowest two levels (0.025 and 0.080 mg/l) on Day 14 were low, 64 and 61% of nominal respectively; at the remaining levels (0.25 and 0.8 mg/l) measured levels were 109 and 104% of the nominal value after 14 days. In addition, although the measured levels in the freshly prepared test media on Day 21 ranged from 92 to 106% on the nominal values, the measured levels in the expired (24 hours old) media were low, ranging from 19 to 51% of nominal. The decrease in the measured levels was attributed to microbial growth in the test vessels and subsequent biodegradation of the test item.
Details on test conditions:
The study employed 10 replicate vessels in each control and test group, each containing a single daphnid and 80 mL of medium in 100 mL glass beakers.
Daphnia were exposed to the prepared control and test media for a period of 21 days under semi-static exposure conditions with batchwise renewal of the media in each vessel after every 48 hours.
The test was conducted in a temperature-controlled laboratory at nominally 20 ± 1°C with a photoperiod of 16 hours light : 8 hours dark. The light intensity of the test area was measured to be < 800 Lux. The culture medium was not aerated for the duration of the study.
In accordance with the test guideline, Daphnia were fed with a suspension of the green alga Chlorella vulgaris at a rate of 0.15 mg C per Daphnia per day.
Reference substance (positive control):
21 d
Dose descriptor:
Effect conc.:
0.254 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
21 d
Dose descriptor:
Effect conc.:
0.831 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
Details on results:
The mortality of the parental Daphnia in the control and solvent control group was 0 and 10% respectively and fecundity in the controls was 108.4 and 152.1 individuals per adult, confirming the validity of the test.

Cumulative numbers of juveniles produced per adult alive for 21 days in each test vessel and results of statistical comparison ot the mean values (by Dunnett's multicomparison test).

 Vessel No.                    Nominal concentraion (mg/L) (Measured concentration TWA, mg/L)
   Control  Solv. Control  0.025 (0.023)  0.080 (0.071) 0.250 (0.254)  0.80 (0.831) 2.50 (2.62) 
 1  110 155  146  109  147  122 
 2  102 154  157  156  125  119 
 3  145 133  164  155 126 
 4  88 130  153  161  148  125 
 5  112 156  157  148  149  143 
 6  107 150  115  114  142  114 
 7  102 172  148  152  145  119 
 8  113 153  181  143  142  125 
 9  OL 170  155  163  142  120 
 10  97 129  136  165  123  121 
 Mean  108.4 152.1  148.1  147.5  141.8  123.4  0.0 
 S.D.  15.8 14.9  17.6  20.3  10.2  7.8   
 Inhibition rate (%)     2.6  3.0  6.8  18.9  100.0 
 Significant difference     **  ++ 

D: Were not included for calculation because the parental Daphnia was dead during a 21d testing period.

OL: Rejected as outlier

-: Indicates no significant difference

**: Indicates a significatn difference (alpha = 0.01) from the solvent control

++: Statistical comparison test could not be performed for this concentration because no adult was alive after 21 d. However, it was concluded that this concentration level showed adverse effect on Daphnia reproduction.

Validity criteria fulfilled:
The mortality of the parental Daphnia in the control and solvent control group was 0 and 10% respectively and fecundity in the controls was 108.4 and 152.1 individuals per adult, confirming the validity of the test.
Executive summary:

The test has been conducted according to OECD Guideline 211 (Daphnia magna Reproduction test) under semi-static conditions for 21d. 5 concentrations were tested (0.025, 0.080, 0.250, 0.800, 2.50 mg/L). As the solubility of TPP is low, a vehicle has been used (HCO-60:dimethylformamide 1:1 (v/v)). 10 animals were tested per concentration each in a vessel and the number of juveniles was counted. The substance concentration was followed during the test. Since there were analysis results exceeding ± 20%, the calculation of the inhibitory concentration was carried out using a time weighted average.

A NOEC of 0.254 mg/L and a LOEC of 0.831 were found in this study regarding the reproduction of Daphnia magna.

Description of key information

The chronic toxicity of TPP to aquatic invertebrates (Dapnia magna) was tested according to OECD Guideline 211 (Daphnia magna Reproduction test). After 21 days a NOEC of 0.254 mg/L was obtained.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.254 mg/L

Additional information

In a study according OECD 211 the long-term toxicity of thriphenyl phosphate to daphnia magna was assessed (Baumann 2014). The determined NOEC was 52 µg/L and the EC10 was 120 µg/L. However, the information in the published assessment by the Bavarian authorities lack many details, especially the basis for the effect concentrations. Therefore, the reliability of the results has to be assigned as Klimisch 4 and the results are not used for further evaluations. Furthermore, the relevant effec concentrations are in the same order of magnitude and variations might be the result of different reasons, e.g. interlaboratory variations. In addition, invertebrates are not the most sensitive species and PNEC derivation will rely on the results of long-term toxicity to fish.