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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Adequacy of study:
other information
Test organisms (species):
Daphnia magna
Test type:
semi-static
Total exposure duration:
21 d
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
3 mg/L
Nominal / measured:
nominal
Basis for effect:
reproduction
Executive summary:

The study (Hüls 1994) established NOEC and LOEC values for DIBP concerning long-term effects on daphnia magna.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Conclusions:
Measured effect concentrations [mg/l]:

NOEC (10d): 0.10 (Gammarus pulex)
NOEC (16d): 0.56 (Daphnia magna)
NOEC (21d): 1.00 (Daphnia magna)

EC50 (7d): 0.54 (Dugesia japonica)
EC50 (21d): 1.05 ((Daphnia magna)
Executive summary:

Long-term toxicity of DBP foraquatic invertebrates

 

No.

Species

Result(mg/l)

Method

Reference

1

Daphnia magna

1(21dNOEC)

Other

Kuhn et al.(1989)

2

Daphnia magna

0.56(16dNOEC)

OECD202

McCarthy and Whitmore (1985)

3

Daphnia magna

1.05 (21 dEC50)

EPA600/8-87/011

De Foe et al.(1990)

4

Dugesia japonica

0.54 (7 dEC50)

Other

Yoshioka et al.(1986)

5

Gammarus pulex

0.10(10dNOEC)

Other,flowthrough

Thurén and Woin (1991)

 

 

The IPCS document on DBP (IPCS/WHO,1997) contains some other long-term toxicity studies with aquatic invertebrates. The effect concentrations in these tests were all larger than100 µg/l.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well documented and scientifically acceptable, but lacking guidelines
Test organisms (species):
Daphnia magna
Details on test organisms:
The Daphnia magna strain (IRCHA strain) has been maintained in accordance with the procedure practised since 1978. In each case, 20-30 specimens were placed in forty 2-l. beakers which had been filled with at least 1.6l. Berlin tap water. They provided 24 h-old animals when the offspring
were removed daily from the cultures.
For all Daphnia strain cultures, temperature-controlled, dechlorinated and oxygen-saturated tap water (German hardness 16 °, pH value 7.6-7.7) was used which had been left to stand for 24 h. Before collecting the water, the tap was turned on fully and left to run for at least 1 h.
All beakers were covered with watch glasses and placed on a white supporting surface. Feeding with dry algae of the Scenedesmus genus took place daily. Nine g of feed were suspended in 1000 ml tap water and 2 ml of the suspension were added to each beaker.
The temperature of the culture area was regulated thermostatically at 20°C. Under exclusion of daylight, the area was lit by fluorescent lamps (Philips TL 65/33W) for 9 h between 7 a.m. and 4 p.m.
On Monday and Thursday of each week the tap water in all beakers was renewed as were the beakers themselves on Mondays. On Mondays, the offspring which had appeared between Thursday or Friday and Monday were concentrated using the 0.315 mm DIN sieve and separated according
to size using the 0.630 mm DIN sieve. Daphnia in the different size categories were used separately for further cultivation.
In order to obtain 24 h-old animals on the potential preparation days in a 21 d test series - Wednesdays or Fridays- it was necessary to remove the offspring from the cultivation beakers on Tuesday and/or Thursday. The daphnids which were at most 24 h old were removed by pipette and concentrated on a 0.25 mm DIN sieve, placed in as small an amount of dilution water as possible and used as test organisms.
Test type:
semi-static
Water media type:
freshwater
Total exposure duration:
21 d
Remarks on exposure duration:
also preliminary 24h acute test
pH:
8.0 +/-0.2
Details on test conditions:
In the interests of national and international standardization, an artificial medium (synthetic fresh water) (DIN - German Institute of Standardization, 1982) of the following composition was used in the test and control preparations:
11 .76 g CaCl2. 2H20 (A.R.)/I litre deionized water
4.93 g MgSO4. 7H20 (A.R.)/I litre deionized water
2.59 g NaHCO3 (A.R.)/1 litre deionized water
0.23 g KCl (A.R.)/1 litre deionized water.

Twenty-five millilitres of each solution was pipetted into a graduated flask and completed to I litre with deionized water. The amount of calcium and magnesium ions in this solution was 2.5mmol l^-1. The molar relationship of sodium to potassium ions was 10:1. This water was aerated up to the water saturation level and the pH value was measured (8.0 + 0.2). When using deionized water with a conductivity of < 1µS cm^-1, the dilution water was diluted with 10% tap water.
Reference substance (positive control):
yes
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
8.9 mg/L
Nominal / measured:
nominal
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
17 mg/L
Nominal / measured:
nominal
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Basis for effect:
mortality

In order to facilitate comparison of the results, the following data were included in Table 1 on the substances listed according to substance group:

The 24 h EC0 and the 24 h EC50 (referred to the nominal value) for the acute Daphnia test.

The NOEC as referred to the nominal value and, in addition, the minimum value of the test concentration range, the dilution ratio, the

most sensitive parameter and the type of vessel used in the 21 d Daphnia reproduction test.

From data on the NOEC and the dilution ratios, it was possible to identify the lowest concentration tested where an effect of the substance could be observed.

Table 2 lists the substances according to their harmful effects (as referred to the nominal value) beginning with the most toxic. The minimum value was also given.

Table 2 reveals a higher toxicity of phthalates with increasing alkyl chain length. In comparison with phthalic acid diethyl ester, the NOEC of phthalic acid diallyl ester was 4 times lower; it was 13 times lower in the case of phthalic acid dibutyl ester.

Table 3 shows the nominal concentrations obtained for the 24 h EC50 and the 21 d NOEC by substance groups. The nominal concentrations had to be given as no results of chemical analysis were available for the 24 h EC50. For each tested substance, the statistically confirmed 24h EC50 and NOEC values were related to each other whereby a substance concentration of NOEC = l was used.

Conclusions:
The 24h EC0 for DBP was 8.9 mg/l and the EC50 was 17 mg/l. The 21d NOEC to reproduction was 1.0 mg/l nominal value and 0.5 mg/l minimum value. The most sensitive parameter was parent mortality.
Executive summary:

The study tested the effects of 73 substances on daphnia magna. Only those related to DBP are documented here.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well documented and scientifically acceptable, but lacking guidelines
Principles of method if other than guideline:
No guideline specified, cf. "Any other information on material and methods" for details.
GLP compliance:
not specified
Details on test solutions:
Each of the three phthalates was dissolved in Mallinckrodt Nanograde acetone and the proper quantity of this acetone stock solution was dissolved in artificial seawater to prepare exposure media of the concentrations listed above. Two acetone stock solutions with different phthalate concentrations were used so that no more than 1 ml of acetone /l would be added to any exposure medium. This concentration of acetone has been found to be completely non-toxic to crustacean larvae. The control cultures (0 ppb phthalate, nominal) received 1 ml of acetone /l of seawater.
Test organisms (species):
Palaemonetes pugio
Details on test organisms:
Gravid female Palaemonetes pugio were collected from salt marshes at the eastern end of Galveston Island, Texas. Separate collections were made between June and October, 1976, for testing each phthalate ester. Previous observations have established that the overall health and viability of adults do not vary significantly during this time of year (Tatem et al., 1976). In the laboratory, the gravid females were maintained in 100 liter fiberglass tanks containing artificial seawater at a salinity (S) of 15 per mille and a temperature of approximately 22°C. A nylon mesh screen with a mesh size large enough to allow the larvae but not the adults to pass through, was placed near one end of the tank. Newly hatched larvae were attracted through the screen by a light placed beyond the divider. Daily as needed, the larvae were removed for use and the remaining gravid females were moved to a similar tank with new seawater. By using this procedure we were assured that all larvae used were less than one day old. While it was highly unlikely that one day's hatch consisted of the progeny of only one female, zoeae obtained on any day were from a limited number of females and these were different each day.
Test type:
semi-static
Water media type:
saltwater
Total exposure duration:
37 d
Test temperature:
22 +/-1° C
Reference substance (positive control):
yes
Reported statistics and error estimates:
In most cases mean initial concentrations were similar to the nominal values. However at the highest concentration of DBP and the 3 highest concentrations of DEHP, mean initial measured concentrations were significantly lower than the nominal values. At the highest concentrations of DBP
and DEHP, small droplets of phthalate were sometimes observed in the exposure medium indicating incomplete equilibration. This probably accounts for the large standard deviations observed for some measured concentrations. The 24 h phthalate concentrations were always lower than the initial values, indicating loss of phthalates from the test mixtures between media changes. Thus, the actual mean exposure concentrations were always lower than the initial nominal values.

Toxicity

DBP was substantially more toxic to Palaemonetes larvae than DMP (Figure 2). At 10 and 50 ppm DBP, there was 100°70 mortality in 3 to 4 days. However, at these nominal concentrations, fine droplets of DBP were observed to coalesce from the medium

and remain in suspension or migrate to the surface. Larvae encountering such droplets might become coated and thus receive a greater than expected dose of DBP. Control survival was significantly lower, 51%, than in the DMP experiment, indicating inter-hatch variability in the viability of the larvae. At exposure concentrations of 100, 500 and 1000 ppb survival was 45, 64 and 41%, respectively.

A highly significant decrease in survival resulted from exposure to DMP and DBP (Table II). However, as indicated above, mortalities observed at 10 and 50 ppm DBP may not have been due to chemical toxicity of phthalate in solution. When data from these 2 DBP concentrations are excluded then DBP exposure did not have a significant effect on larval survival (p > F = 0.4).

Molting Rate

Larvae exposed to DBP showed the same intermolt pattern as those exposed to low concentrations of DMP. There was some suggestion of molt desynchronization for animals exposed to 1 ppm DBP. This effect was not as obvious as that seen in animals exposed to the highest concentrations of DMP. No effect was apparent until after the first zoeal molt (i.e., after day 4). In no case was there evidence for any phthalate of a dose-dependent change in the stage structure of zoeal development.

Duration of larval development

The mean duration of zoeal development of larvae exposed to the 3 phthalates is shown in Figure 4. At DMP concentrations between 100 ppb and 10 ppm, the mean duration of development from hatching to the first postlarval instar was slightly less than that for the controls. At 100 ppm DMP, the mean duration of zoeal development was nearly twice as long as that of the controls. The effects of DBP and DEHP on developmental rate were more variable. Exposure concentrations of 100 and 500 ppb DBP resulted in mean developmental rates lower and higher, respectively, than the control rate. However, at 1 ppm DBP, developmental rate was the same as that of the controls. There was little variation from control values in the developmental rate of larvae exposed to any concentration of DEHP.

The regression analysis of the molting rate data is shown in Table III. Once again, exposure data for each phthalate ester were analyzed separately. Each hatch was considered a block. Larvae exposed to DMP showed highly significant differences in response to both treatment and hatch of the larvae. The interaction of the treatment and source of larvae (block) was significant.

Animals exposed to DBP showed no significant difference in response due either to treatment or hatch. The interaction of treatment and hatch was significant, suggesting that the differences observed were due to the extreme variability of different exposure groups. The correlation for this data was poor (r= 0.304), suggesting that uncontrolled factors, not DBP exposure, produced the significant

differences among groups.

Conclusions:
Doses of 10 or 50 ppm DBP caused 100% mortality within 3 to 4 days. Slighter doses caused almost no more mortality than within the control group (which was extraordinary high!, ~45%), but some developmental effects (duration of larval development) were recorded.
Executive summary:

The effects of three phthalic acid esters, dimethyl phthalate (DMP), di-n-butyl phthalate (DBP) and di-2-ethylhexyl phthalate (DEHP) on survival and development rate of larvae of the grass shrimp Palaemonetespugio were investigated. Only 100 ppm DMP and 10 to 50 ppm DBP were acutely toxic to the larvae.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guidelines
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
other: OECD No. 202 (Daphnia sp. acute immobilization test and reproduction test)
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
not specified
Analytical monitoring:
yes
Details on test solutions:
The phthalate ester was dissolved in acetone, used as a carrier for its introduction into test exposure water. In all tests, two controls were run: an uncontaminated water control (no toxicant or carrier, designated “control” in the figures and tables) and a carrier control containing acetone at the same concentration as in the toxicant exposures (designated “0.0” in the figures and tables). Water quality characteristics of the dechlorinated tap water used during the experiments are listed in Table 1.
Test organisms (species):
Daphnia magna
Details on test organisms:
The stock of D. magna used in these tests was procured from Miami University (Ohio) and has been maintained in our laboratory since April 1977. The D. magna stock was maintained on a diet of Purina Trout Chow and yeast. Test animals for both the acute immobilization tests (rangefinding
test) and the reproduction test were collected when the animals were less than 24 h old. The range-finding tests were run for 48 h; the reproduction tests were run for 15 to 16 d, which was the time required for production of at least three broods.
Test type:
semi-static
Water media type:
freshwater
Total exposure duration:
16 d
Test temperature:
21°C
pH:
7.9-8.1
Dissolved oxygen:
5.8-6.9
Nominal and measured concentrations:
nominal: 0, 0.056, 0.18, 0.56, 1.8, 3.2 mg/l
Reference substance (positive control):
yes
Duration:
48 d
Dose descriptor:
LC50
Effect conc.:
4.7 - 5.6 mg/L
Nominal / measured:
nominal
Remarks on result:
other: 95% CL, range-finding acute mortality test
Duration:
16 d
Dose descriptor:
NOEC
Effect conc.:
0.56 mg/L
Nominal / measured:
nominal
Basis for effect:
reproduction
Duration:
16 d
Dose descriptor:
LOEC
Effect conc.:
1.8 mg/L
Nominal / measured:
nominal
Basis for effect:
reproduction

In the acute mortality test (range-finding test), all D. magna were dead after 48 h of exposure to nominal concentrations of 7.5

and 10.0 mg/L DBP. At the lower doses of 3.0, 1.0 and 0.5 mg/L DBP and in controls, all animals survived, except for one individual at 3.0 mg/L. The LC50 (lethal concentration to 50% of the test population) is between 3.0 and 7.5 mg/L DBP. Although a probit analysis cannot be performed, because this procedure requires two responses that are between 0 and 100% mortality, a nonparametric analysis was developed for steep dose-response bioassays (Schmoyer, Beauchamp and McCarthy, manuscript in preparation). The LC50 was estimated using this method and was equal to 5.2 mg/L, with 95% confidence limits of 4.7 and 5.6

mg/L.

Temperature, DO and pH remained relatively constant throughout each test and across all concentrations (Table 2). The measured concentrations of DBP in the freshly prepared solutions (before animals were added) were fairly close to the nominal doses; however, after 24 h, the concentrations dropped by about one-third (Table 2).

The results of the filtration experiment designed to determine the fraction of DBP bound to the suspended particulates in the food demonstrated that only 2.5% (*0.2%) of the DBP was bound to the suspended particulate matter.

Survival of D. magna exposed to DBP exceeded 80% in all concentrations except 1.8 and 3.2 mg/L and in the carrier-free control. The reason for the poor survival (and poor reproduction) of the control group is not clear. By the end of the experiment (day 16),

70% of the D. magna were alive at 1.8 mg/L and 18% were alive at 3.2 mg/L DBP (Fig. 1 and Table 3).

The number of young produced per surviving adult at each dose is indicated in Table 3. Reproduction in D. magna was stimulated at low levels and inhibited at higher concentrations of DBP. Exposure to 0.056 to 0.56 mg/L DBP increased the total numbers of young produced. Reproduction was significantly impaired at 1.8 mg/L, compared with that at either the 0.56 mg/L dose or the carrier control. Although eggs were occasionally observed in the brood sacs of animals exposed to a dose of 3.2 mg/L DBP, no viable

young were produced. If the no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) are assumed to reflect only inhibition of reproduction (as opposed to the apparent stimulation observed at moderate doses), then the NOEC for DBP is 0.56 mg/L and the LOEC is 1.8 mg/L (nominal concentrations).

Since the animals exposed to DBP were cultured individually, information was obtained on the effect of DBP on the total number of broods (as defined by appearance of eggs in the brood sac, rather than by release of viable young) and on the number of days and number of molts required to achieve reproductive maturity (primiparous instar). DBP had relatively little effect on any of these

parameters, although the highest dose did result in a significant decrease in the total number of broods and in the instar at which eggs were first observed in the brood sac (primiparous instar, Table 4).

Validity criteria fulfilled:
yes
Conclusions:
For D. magna, exposure to 1.8 mg/L DBP caused a significant reduction in reproduction. Doses of 0.56 mg/L DBP had no significant effect in decreasing reproduction.
Executive summary:

The toxicities of di-n-butyl phthalate (DBP) and di-n-octyl phthalate (DOP) were assessed by measuring the effect of exposure to these compounds on the fecundity of Daphnia magna and on the hatching and survival of the early life stages of the fathead minnow Pimephales promelas. Only the effects on Daphnia magna concerning DBP are documented here.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Specific details on test material used for the study:
All of the chemicals were colorless liquids with purities >95%. The aqueous solubilities of these chemicals were reported previously and are summarized as follows (mg/L): DBP (11.2).
Analytical monitoring:
yes
Test organisms (species):
Daphnia magna
Details on test organisms:
The test species were waterfleas (Daphnia magna). Full life-cycle (21 d) studies were performed with D. magna. The D. magna (<=24 h) were used to initiate the chronic tests and were acclimated to the appropriate temperature before being tested.
Test type:
flow-through
Water media type:
freshwater
Total exposure duration:
21 d
Hardness:
150-180 mg/l
Test temperature:
21° +/-2° C
pH:
7.9-8.3
Dissolved oxygen:
>60%
Conductivity:
400-600 µmho/cm
Reference substance (positive control):
yes
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.96 mg/L
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
2.5 mg/L

Geometric mean maximum acceptable toxicant concentrations (GM-MATC) were determined for all 14 phthalate esters except DUP (Table 3), where no effects on survival or reproduction were observed even at the highest measured concentration (0.059 mg/L). The GM-MATC values ranged from 0.042 mg/L for DINP to 38.4 mg/L for DEP (Table 4). For DMP, DEHP, 71 lP, and DTDP, survival was the most sensitive end point. For the other 10 phthalate esters, survival and reproduction were equally sensitive. Test-to-test variation in control daphnid reproduction (mean offspring per adult female) ranged from 56 to 116; however, it meets ASTM E-I193 minimum criteria for an acceptable test. Variation between replicate test chambers and treatment levels was much less than between tests and allowed for sufficient statistical power to evaluate potential reproductive effects. Test-to-test variation in control offspring reproduction is of secondary importance relative to variation between replicates and treatment levels.

The GM-MATC tended to decrease as the molecular weight and alkyl chain length increased, up to a chain length of 4 to 6 carbon atoms. This trend has been reported for acute toxicity studies for these same esters with multiple species. One might expect, a priori, that toxicity would increase and the water solubility decrease as the alkyl chain length increases. This does not appear to be true. Phthalate

esters with alkyl chain lengths >= 6 carbon atoms have measured water solubility values in the range of 0.09 to 1.2 mg/L, and they do not decrease linearly with alkyl chain length. This is possibly due to micelle formation. Phthalate ester molecules have both a polar and a nonpolar group that lends itself to the formation of micelles. Micelles are formed, due to coalescence of the test chemical, when the critical micelle concentration is exceeded. This results in the formation of microdroplets of pure chemical that are entrained in water during the preparation of solutions for water solubility measurements

and toxicity tests. As a result, operationally defined water solubility values are obtained since the standard phase separation techniques do not provide separation of the micelles/microdroplets from the water solution. This explains the lack of correlation between water solubility and alkyl chain length and suggests that exposure concentrations used in the present test, a well as in other previously reported toxicity

tests, may be greater than the “true” water solubility of the test chemicals.

A comparison between the acute LC50 value and the GMMATC (acute-to-chronic ratio; ACR) was possible for two of the 14 phthalate esters. The ACR values for DEP and DBP were calculated using acute LC50 values previously reported. The values were very small, 2.2 and 2.3, respectively. It was not possible to calculate ACRs for phthalate esters with a molecular weight greater than DBP because

these phthalate esters were not acutely toxic at concentrations approximating their aqueous solubility.

Conclusions:
The measured NOEC was 0.96 mg/l and the LOEC 2.5 mg/l.
Executive summary:

Chronic toxicity studies were performed with commercial phthalate esters and Daphnia magna (14 phthalates) and rainbow trout (Oncorhynchus mykiss) (six phthalates). Only the experiments on the daphnia magna concerning DBP are documented here.

Description of key information

Measured effect concentrations [mg/l]:
NOEC (10d): 0.10 (Gammarus pulex)
NOEC (16d): 0.56 (Daphnia magna)
NOEC (21d): 1.00 (Daphnia magna)
EC50 (7d): 0.54 (Dugesia japonica)
EC50 (21d): 1.05 ((Daphnia magna)
For the chemical safety assessment the most sensitive endpoint has been chosen:

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
0.1 mg/L

Additional information