L-Valine, N-(1-oxopentyl)-N-[[2'-(2H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]-, phenylmethyl ester

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:

Feb - July 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

The day before the start of the study, singular samples were taken from the lowest and the highest test concentration. All test concentrations and the solvent-control were analysed in duplicate at the start of the study. In addition, a singular sample was taken from the 1.4 mg/ml stock, and following spiking of the medium, extra samples were taken from the 0.14 mg/1 solution, since the test concentration in this solution was too low. On day 0, two samples for TOC analysis were taken from the test medium (blank-control).
During the study duplicate samples were taken from all concentrations and the solvent control.
Sampling Frequency On day 7, 14 and 21.
Sample volume 10ml

Vehicle:

yes

Remarks:

methanol, M7

Details on test solutions:

The test media were prepared using stock solutions with nominal concentrations of 0.6, 1.4, 3.2, 6.0 and 14 mg/ml in methanol, where the stock at 0.6 mg/ml was a dilution in methanol of the stock at 6.0 mg/ml. The stock solutions were clear and colourless. In the flow-through system the dilution water (M7 medium) was dosed separately from the test substance stock solutions into mixing vessels. The dosed volumes and the dilution water were mixed under continuous stirring in the mixing vessels before entering the test vessels. The dosing was computer controlled, and the system was checked daily.

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.

Test type:

flow-through

Water media type:

freshwater

Total exposure duration:

21 d

Hardness:

>140 mg/I

Test temperature:

21.4 and 22.1 °C

pH:

6.0 to 9.0

Dissolved oxygen:

> 3.0 mg/I

Nominal and measured concentrations:

Analytical results of the samples taken in the Daphnia magna, reproduction test with PBS859DS.
Concentrations of PBS 859 DS in mg/I

target day 0 day 7 day 14 day 21 average % of target
0.06 0.0609 0.0575 0.0504 0.0465 0.054 90
0.14 0.157* 0.121 0.131 0.120 0.132 95
0.32 0.296 0.265 0.245 0.285 0.273 85
0.6 0.511 0.476 0.460 0.615 0.515 86
1.4 1.30 1.19 1.18 1.12 1.20 85
* This concentration was achieved after sp1k1ng of the test medium, which was necessary since the initial measured concentration was too low

Reference substance (positive control):

no

Remarks:

Control: Test medium without test substance or other additives (O mg/I) and one control containing test medium with the additive (methanol) used in the treatment of the stock solutions.

Key result

Duration:

21 d

Dose descriptor:

EC50

Remarks:

for parental survival

Effect conc.:

0.74 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

21 d

Dose descriptor:

EC50

Remarks:

Debtox

Effect conc.:

0.65 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.6 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.14 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

body length

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

body length

Details on results:

LIST OF DEVIATIONS TO THE PROTOCOL
1. The blank-control was dosed via an HPLC pump (Waters) instead of by a diluter or 8-way valve
(Gilson). This did not affect the study, since the flow of the dosed medium was similar to that of
the other test vessels. ·
2. Light intensity was somewhat higher than prescribed by the protocol (600-820 lux instead of 600-
800 lux). This deviation is rather small and is not considered to have affected the test, since the
acceptability criteria were met.
3. On day 0, test conditions were not measured in the blank-control. This did not affect the study,
since no differences were recorded in daphnia condition as compared to the solvent-control.
4. The lowest stock solution was not prepared separately, but was a dilution of the 10-fold higher
stock. This did not affect the study, since analyses showed that the target concentration was
reached.
5. On day -1, singular instead of duplicate samples were taken for analysis. This did not affect the
study, since sufficient samples were taken at day O, showing that the actual concentrations were
in agreement with target concentrations.
6. On day 14, 20 and 21, the oxygen concentration was measured in all concentrations, since the
difference between the blank-control and the solvent-control was 1.7 mg/I on day 14 and the
daphnids showed a white brood pouch on day 20. This deviation resulted in more information and
therefore did not negatively influence the study.
7. Since on day 20, the oxygen concentration was below 3 mg/I in several vessels, aeration was
introduced in the mixing vessels. The study integrity was not affected by this deviation, since it
resulted in an increase of the oxygen concentration which implied that the study met the acceptability criteria.
8. Results were based on target concentrations instead of on average exposure concentrations.
This was not considered to affect the study result, since analyses showed that measured
concentrations were all above 80% of the corresponding target concentrations.

Mortality of the parental daphnids in the solvent-control did not exceed 20% at the end of the test.

Although mortality in the blank-control exceeded 20%, this was not considered to have affected the

test, since it could be attributed to a mechanical failure in the dosing system rather than to an intrinsic

higher mortality rate of the daphnids.

The mean number of living offspring per control parent at the end of the test was >60 (91 ± 19 and 147

± 9 in the blank-and solvent-control, respectively). The test conditions remained within the ranges

prescribed by the protocol with the following exceptions. A small deviation in pH was observed at the

highest concentration at the end of the test: this value differed by 1.6 units from the initial pH value.

This decrease might be due to the presence of a lower number of young daphnids as compared to the

controls, resulting in an excess of algal feed. The oxygen concentration in all but one of the test

concentrations was below 3 mg/I on day 20. This was suspected to be caused by yeast observed in

the brood pouches of the daphnids on this day. The deviation was believed to have only occurred for a

short period of time. This is reinforced by the observation that both reproduction and mortality were

rather constant over the entire test period; thus, the decrease in oxygen concentration was not

considered to have affected these parameters. Moreover, there were no signs of oxygen stress, and

therefore, this deviation was not considered to have affected the test. The test vessels were replaced

by clean vessels and aeration was introduced in the mixing vessels and only turned off at the end of

the test. The next day, the oxygen concentrations were well above 3.0 mg/I. Finally, the coefficient of

variation around the mean number of offspring per parent at the end of the test was just above 20%

(21 %) in the blank-control, but well below 20% (9%) for the solvent-control. The higher variation in the

blank-control was probably due to the mechanical failure of the dosing system in this group. The study

was considered to be valid according to the validity criteria described in the protocol.

Survival decreased with increasing concentration, with no daphnids surviving at the highest

concentration tested. The increase was closely related to concentration, with the exception for the

mortality at 0.14 and 0.32 mg/I, which did not completely follow this trend.

Statistical analysis of the reproduction data revealed that the reproductive capacity of daphnids

exposed to PBS 859 DS up to 0.32 mg/I was not negatively affected. A significant decrease in

reproductive capacity was found at the target concentration of 0.60 mg/I and above (Dunett test,

Bonferroni t-test and Tukey test: a=0.05).

Body lengths were significantly reduced at 0.06, 0.32 and 0.60 mg/I, but not at 0.14 mg/I (Bonferroni ttest

and Tukey test, a=0.05). Since the reduction at 0.06 mg/I was only 5%, and there was no

statistically significant effect at 0.14 mg/I, the NOEC for body length was set at 0.14 mg/I, and the

LOEC for body length was set at 0.32 mg/I.

No other concentration related effects were observed in the target concentrations up to 0.60 mg/I

during the test, while effects on mobility and condition were observed at 1 .4 mg/I. At day 20, daphnids

at all target concentrations had a white brood pouch. This phenomenon did not invalidate the test,

since it did not affect the daphnids in their survival nor reproduction.

Validity criteria fulfilled:

yes

Conclusions:

Parameter PBS 859 DS (mg/I)

Observed 21-day EC100 for parental survival 1.4
Calculated 21-day ECso for parental survival* 0.74
Observed 21-day ECo for parental survival 0.06
Estimated NEC for parental survival* 0.17
95% Confidence interval of NEC* 0-0.34
21-day ECso for reproduction* 0.65
21-day ECso for reproduction** 0.71
95 % Confidence interval** 0.34-1.5
21-day LOEC for reproduction# 0.60
21-day NOEC for reproduction# 0.32
21-day LOEC for body length## 0.32
21-day NOEC for body length## 0.14
Overall LOEC 0.32
Overall NOEC 0.06
* Debtox
** Linear regression analysis
# ANOVA-Bonferroni t-test, Dunnet t-test and Tukey's test; α =0.05
## ANOVA-Bonferroni t-test and Tukey's test; α =0.05

Description of key information

Parameter                            PBS 859 DS (mg/I)

Observed 21-day EC100 for parental survival       1.4

Calculated 21-day ECso for parental survival*       0.74

Observed 21-day ECo for parental survival       0.06

Estimated NEC for parental survival*              0.17

95% Confidence interval of NEC*              0-0.34

21-day ECso for reproduction*              0.65

21-day ECso for reproduction**              0.71

95 % Confidence interval**              0.34-1.5

21-day LOEC for reproduction#              0.60

21-day NOEC for reproduction#              0.32

21-day LOEC for body length##              0.32

21-day NOEC for body length##              0.14

Overall LOEC                     0.32

Overall NOEC                     0.06

* Debtox

** Linear regression analysis

# ANOVA-Bonferroni t-test, Dunnet t-test and Tukey's test;  α =0.05

## ANOVA-Bonferroni t-test and Tukey's test; α =0.05

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.06 mg/L

Additional information

For the test substance, a chronic daphnia test is available. This GLP study was conducted under flow-through conditions and according to OECD guideline 211. Based on reproduction, a NOEC (21 d) of 0.32 mg/L (nominal) was determined in this test. The overall NOEC (21 d) was reported as 0.06 mg/L (nominal). According to the classification criteria described in REGULATION (EC) No 1272/2008 (CLP) the substance has to be classified as chronic aquatic toxic, cat 1.